Stage- and Rearing-Dependent Metabolomics Profiling of Ophiocordyceps sinensis and Its Pipeline Products.

Cordyceps, a parasitic complex of the fungus Ophiocordyceps sinensis (Berk.) (Hypocreales: Ophiocordycipitaceae) and the ghost moth Thitarodes (Lepidoptera: Hepialidae), is a historical ethnopharmacological commodity in China. Recently, artificial cultivation of Chinese cordyceps has been established to supplement the dwindling natural resources. However, much is unknown between the natural and cultivated products in terms of nutritional aspect, which may provide essential information for quality evaluation. The current study aims to determine the metabolic profiles of 17 treatments from 3 sample groups including O. sinensis fungus, Thitarodes insect and cordyceps complex, using Gas Chromatography - Quadrupole Time-of-Flight Mass Spectrometry. A total of 98 metabolites were detected, with 90 of them varying in concentrations among groups. The tested groups could be separated, except that fungal fruiting body was clustered into the same group as Chinese cordyceps. The main distinguishing factors for the groups studied were the 24 metabolites involved in numerous different metabolic pathways. In conclusion, metabolomics of O. sinensis and its related products were determined mainly by the fruiting bodies other than culture methods. Our results suggest that artificially cultured fruiting bodies and cordyceps may share indistinguishable metabolic functions as the natural ones.

Discovery of the signal pathways and major bioactive compounds responsible for the anti-hypoxia effect of Chinese cordyceps.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypoxia will cause an increase in the rate of fatigue and aging. Chinese cordyceps, a parasitic Thitarodes insect-Ophiocordyceps sinensis fungus complex in the Qinghai-Tibet Plateau, has long been used to ameliorate human conditions associated with aging and senescence, it is principally applied to treat fatigue, night sweating and other symptoms related to aging, and it may play the anti-aging and anti-fatigue effect by improving the body's hypoxia tolerance. AIMS OF THE STUDY: The present study investigated the anti-hypoxia activity of Chinese cordyceps and explore the main corresponding signal pathways and bioactive compounds. MATERIALS AND METHODS: In this study, network pharmacology analysis, molecular docking, cell and whole pharmacodynamic experiments were hired to study the major signal pathways and the bioactive compounds of Chinese cordyceps for anti-hypoxia activity. RESULTS: 17 pathways which Chinese cordyceps acted on seemed to be related to the anti-hypoxia effect, and "VEGF signal pathway" was one of the most important pathway. Chinese cordyceps improved the survival rate and regulated the targets related VEGF signal pathway of H9C2 cells under hypoxia, and also had significant anti-hypoxia effects to mice. Chorioallantoic membrane model experiment showed that Chinese cordyceps and the main constituents of (9Z,12Z)-octadeca-9,12-dienoic acid and cerevisterol had significant angiogenic activity in hypoxia condition. CONCLUSION: Based on the results of network pharmacology and molecular docking analysis, cell and whole pharmacodynamic experiments, promoting angiogenesis by regulating VEGF signal pathway might be one of the mechanisms of anti-hypoxia effect of Chinese cordyceps, (9Z, 12Z)-octadeca-9,12-dienoic acid and cerevisterol were considered as the major anti-hypoxia bioactive compounds in Chinese cordyceps.

Toxicological safety evaluation of the cultivated Chinese cordyceps.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese cordyceps, a parasitic Thitarodes insect-Ophiocordyceps sinensis fungus complex in the Qinghai-Tibet Plateau, is one of the most valuable traditional Chinese medicines and health food for ameliorating conditions associated with aging and for treating fatigue, night sweats, hyperglycemia, hyperlipidemia, respiratory, renal and liver diseases, and hyposexuality. The natural Chinese cordyceps resource is rare due to its harsh growing environment, limited geographical distribution and global climate warming. Artificial cultivation of Chinese cordyceps has been successfully established to meet its high demand in market. AIMS OF THE STUDY: The present study aims to evaluate the toxicological safety of the cultivated Chinese cordyceps and provide scientific data for subsequent development and utilization of this valuable biological resource. MATERIALS AND METHODS: The Chinese cordyceps was cultivated by mimicking the habitat environment in low-altitude areas and identified by morphological and microscopic characteristics. Its phytochemical profile was determined by the HPLC. Toxicological studies based on the cultivated Chinese cordyceps were conducted, including chromosomal aberration test of Chinese hamster lung (CHL) cells, Ames test, acute toxicity test and micronucleus (MN) test of bone marrow cells. RESULTS: The Chinese cordyceps successfully cultivated in low-altitude areas exhibited the same morphological and microscopic characteristics as natural Chinese cordyceps. The adenosine content was in accordance with the Chinese Pharmacopoeia (2015 Edition). The HPLC fingerprint was determined and five main chromatographic peaks representing uracil, uridine, inosine, guanosine and adenosine were identified. No dose-dependent increase in the rates of chromosomal aberration was detected in the presence or absence of metabolic activation system. Ames test also demonstrated no dose-dependent increase in the number of reversion mutation for five bacterial strains, with or without rat liver microsomal enzyme mixture (S9) metabolic activation, at a quantity range of 128-5000 µg cultivated Chinese cordyceps per plate. The acute toxicity test with mice showed that after 20 g/kg oral administration of cultivated Chinese cordyceps, neither animal death nor any abnormal change in general dissection of various tissues and organs of the animals were found within 14 days. The median lethal dose (LD50) was greater than 5 g/kg, which is regarded as a non-toxic level, and maximum tolerable dose (MTD) of cultivated Chinese cordyceps in ICR mice was more than 20 g/kg. MN test of mouse bone marrow cells indicated no significant differences among each sample dose and the negative control. CONCLUSION: Based on the results from four toxicological tests, it was concluded that the cultivated Chinese cordyceps was classified as non-toxic in one single administration at high doses by intragastric route in mice. This study provides scientific experimental basis for its safety.

Quorum Sensing Activity and Hyphal Growth by External Stimuli in the Entomopathogenic Fungus Ophiocordyceps sinensis.

The entomopathogenic fungus Ophiocordyceps sinensis is one of the best known and most precious medicines and health food in China. The blastospores-hyphae (dimorphism) transition of this fungus in host hemolymph is critical for the virulence and the mummification of host larvae. To regulate this transition, the effects of inoculum density and fifteen chemicals including fungal nutrients, fungal metabolites, quorum-sensing molecules (QSMs) and insect hormones on the dimorphism in O. sinensis were investigated in vitro. The blastospores tended to exhibit budding growth when inoculated at 107 blastospores per mL, and hyphal growth at concentrations lower than 106 blastospores per mL. At 105 blastospores per mL, the percentage of hyphal formation decreased with the addition of filtered spent medium containing 107 blastospores per mL, indicating the quorum-sensing effect. Blastospores-hyphae transition in this fungus by fifteen chemicals was varied from no response to dimorphic reversion. The addition of N-acetylglucosamine at three concentrations significantly stimulated hyphal formation while inhibiting budding growth. For the first time, insect hormone 20-hydroxyecdysone was found to be involved in the hyphal formation in fungi. These results open new possibilities to regulate the dimorphism, which would be beneficial for the cultivation of the Chinese cordyceps.

Analysis of Volatile Components in Different Ophiocordyceps sinensis and Insect Host Products.

The artificial production of Ophiocordyceps sinensis mycelia and fruiting bodies and the Chinese cordyceps has been established. However, the volatile components from these O. sinensis products are not fully identified. An efficient, convenient, and widely used approach based on headspace solid-phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography and quadrupole time-of-flight mass spectrometry (GC×GC-QTOFMS) was developed for the extraction and the analysis of volatile compounds from three categories of 16 products, including O. sinensis fungus, Thitarodes hosts of O. sinensis, and the Chinese cordyceps. A total of 120 volatile components including 36 alkanes, 25 terpenes, 17 aromatic hydrocarbons, 10 ketones, 5 olefines, 5 alcohols, 3 phenols, and 19 other compounds were identified. The contents of these components varied greatly among the products but alkanes, especially 2,5,6-trimethyldecane, 2,3-dimethylundecane and 2,2,4,4-tetramethyloctane, are the dominant compounds in general. Three categories of volatile compounds were confirmed by partial least squares-discriminant analysis (PLS-DA). This study provided an ideal method for characterizing and distinguishing different O. sinensis and insect hosts-based products.

Identification of the genes involved in growth characters of medicinal fungus Ophiocordyceps sinensis based on Agrobacterium tumefaciens-mediated transformation.

Ophiocordyceps sinensis, one of the well-known and precious fungal species in the world, parasitizes soil-dwelling larvae of ghost moths on the Tibetan Plateau. The genetic intractability of this extremely psychrophilic and slow-growing O. sinensis fungus is a major limitation on the molecular study. In this study, an Agrobacterium tumefaciens-mediated genetic transformation (ATMT) system for this fungus was established. ATMT procedure was optimized based on the fungal recipient, Agrobacterium strains, and different co-cultivation conditions. Blastospores were ideal recipients for this system. Acetosyringone (AS) was not essential for the transformation of O. sinensis. Sixty to 100 hygromycin B-resistant transformants per 1 × 106 blastospores were obtained. Southern blot analysis indicated the presence of a random single-copy integration of T-DNA into the O. sinensis genome. The insertional transformants with altered growth characters such as colony, blastospore, and fruiting body production were selected to identify the T-DNA flanking sequences by modified hiTAIL-PCR and FPNI-PCR techniques. Eight genes, including genes for an MFS transporter, a 2-oxoglutarate dehydrogenase, a DNA-directed RNA polymerase III complex subunit Rpc37, a cytochrome oxidase subunit I, a mitochondrial import inner membrane translocase subunit tim54, a cytidine deaminase, a phosphoribosylaminoimidazole carboxylase, and a histone H3-like centromeric protein cse-4 were identified. This ATMT system provides a useful tool for gene discovery and characterization of O. sinensis and contributes to the better understanding of the mysterious life cycle of O. sinensis and the molecular interaction between this fungus and its host insects.

Comparative Transcriptome Analysis of Thitarodes Armoricanus in Response to the Entomopathogenic Fungi Paecilomyces Hepiali and Ophiocordyceps Sinensis.

Thitarodes armoricanus is a medicinal and economically important Lepidopteran insect species. The larvae infected by Paecilomyces hepiali survive no more than four days, while those infected by Ophiocordyceps sinensis can survive for several months before mummification. This provides a valuable comparative system to study interactions between an insect host and different pathogenic fungi. By using the T. armoricanus genome, a time-course transcriptome analysis of the whole larvae without guts was performed to explore the larvae response to P. hepiali and O. sinensis infection. A total of 3106 differentially expressed genes in five clusters were identified. The genes involved in coagulation and multiple metabolisms were both suppressed after P. hepiali or O. sinensis infection, whereas those related to environmental information responses, cell processes, biotic stimulus, and immunity (such as cecropin (CEC)) were elevated. The rapid death of T. armoricanus after P. hepiali infection might be caused by osmotic imbalance, immunocompromise (such as DEFs and GLVs), and nervous system dysfunction (glutamatergic synapse). Up-regulation of the genes related to cuticle structure, nervous system (such as neurotrophin signal pathway and dopaminergic synapse) and immune effectors (such as attacin (ATT) and proline-rich antimicrobial peptide 1 (PRAMP1)) in T. armoricanus, may contribute to the co-existence of T. armoricanus and O. sinensis. This study provides a global view and potential key genes of the interaction between T. armoricanus and two fungal entomopathogens.

Comparative transcriptome analysis reveals molecular strategies of ghost moth Thitarodes armoricanus in response to hypoxia and anoxia.

Hypoxia or anoxia greatly impact the survival of many animal species. The ghost moth Thitarodes armoricanus is distributed in the Tibetan Plateau at an average elevation of approximate 4 km above sea level and has probably evolved a superior capacity to tolerate low oxygen levels. In this study, transcriptome analysis using high-throughput RNA-seq revealed common and different adaptation strategies of T. armoricanus in response to hypoxia (11% O2) or anoxia. T. armoricanus adopted three common strategies for adaptation to hypoxia or anoxia: Up-regulated signal transduction pathways essential for cellular survival, strengthened cell and organelle structure and activity, and activated immune system. Under hypoxia, T. armoricanus might develop a strategy to adapt to hypoxia by suppressing TCA, oxidative phosphorylation pathways, and hypoxanthine catabolism. T. armoricanus larvae kept active under hypoxia but became coma under anoxia, probably relating to up-regulated or suppressed dopamine synthesis pathway. Furthermore, the HIF system seemed not to be essential for regulating the hypoxic and anoxic responses of this insect in Tibetan Plateau. This study provides a global view of gene expression profiles and suggests common and different adaptation strategies of T. armoricanus under hypoxic and anoxic conditions. The results are helpful for understanding the mechanism responsible for the low oxygen level tolerance of this insect species.

Scratching Stimuli of Mycelia Influence Fruiting Body Production and ROS-Scavenging Gene Expression of Cordyceps militaris.

The entomopathogenic fungus Cordyceps militaris is a valuable medicinal ascomycete, which degenerates frequently during subsequent culture. To avoid economic losses during industrialized production, scratching stimuli of mycelia was introduced to improve the fruiting body production. The present results indicated that higher yields and biological efficiency were obtained from two degenerate strains (YN1-14 and YN2-7) but not from g38 (an insertional mutant in Rhf1 gene with higher yields and shorter growth periods). Furthermore, the growth periods of the fruiting bodies were at least 5 days earlier when the mycelia were scratched before stromata differentiation. Three ROS-scavenging genes including Cu/Zn superoxide dismutase (CmSod1), Glutathione peroxidase (CmGpx), and Catalase A (CmCat A) were isolated and their expression profiles against scratching were determined in degenerate strain YN1-14 and mutant strain g38. At day 5 after scratching, the expression level of CmGpx significantly decreased for strain g38, but that of CmSod1 significantly increased for YN1-14. These results indicated that scratching is an effective way to promote fruiting body production of degenerate strain, which may be related at least with Rhf1 and active oxygen scavenging genes.

Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae).

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the sensitive method to quantify the expression levels of target genes on the basis of endogenous control. An appropriate reference gene set for normalization is essential for reliable results. The ghost moth, Thitarodes armoricanus, a host species of a medicinal fungus, Ophiocordyceps sinensis, is an economically important member of the Lepidoptera. Recent studies have focused on the mechanism of adaptation of this species to its high-altitude environment and host immune response to O. sinensis infection and RT-qPCR is commonly used in these studies to decipher the genetic basis of physiological functions. However, a thorough assessment of candidate reference genes in the genus Thitarodes is lacking. Here, the expression levels of eight candidate reference genes (ACT, EF, EIF4A, GAPDH, G6PDH, RPL13A, TUB and 18S) in T. armoricanus at different developmental stages and in different body parts of the seventh instar larvae were analyzed, along with larvae kept under low temperatures, larvae exposed to two fungal infections and larvae fed different diets. Three established software programs-Bestkeeper, geNorm and NormFinder-were employed to calculate variation among the treatments. The results revealed that the best-suited reference genes differed across the treatments, with EF, EIF4A and GAPDH found to be the best suited for the different developmental stages and larvae body parts; EF, EIF4A and RPL13A found to be the best suited for low-temperature challenge; and EF, EIF4A and TUB found to be the best suited for the fungal infections and dietary treatments. This study thus further contributes to the establishment of an accurate method for normalizing RT-qPCR results for T. armoricanus and serves as a reference for gene expression studies of related insect species.

Photorhabdus luminescens LN2 requires rpoS for nematicidal activity and nematode development.

Photorhabdus (Enterobacteriaceae) bacteria are pathogenic to insects and mutualistic with entomopathogenic Heterorhabditis nematodes. Photorhabdus luminescens subsp. akhurstii LN2, associated with Heterorhabditis indica LN2, shows nematicidal activity against H. bacteriophora H06 infective juveniles (IJs). In the present study, an rpoS mutant of P. luminescens LN2 was generated through allelic exchange to examine the effects of rpoS deletion on the nematicidal activity and nematode development. The results showed that P. luminescens LN2 required rpoS for nematicidal activity against H06 nematodes, normal IJ recovery and development of H. indica LN2, however, not for the bacterial colonization in LN2 and H06 IJs. This provides cues for further understanding the role of rpoS in the mutualistic association between entomopathogenic nematodes and their symbionts.

Identification of the Genes Involved in the Fruiting Body Production and Cordycepin Formation of Cordyceps militaris Fungus.

A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris.

Draft Genome Sequence and Annotation of the Entomopathogenic Bacterium Photorhabdus luminescens LN2, Which Shows Nematicidal Activity against Heterorhabditis bacteriophora H06 Nematodes.

We present here the 5.6-Mb genome sequence of Photorhabdus luminescens strain LN2, a Gram-negative bacterium that is a symbiont of Heterorhabditis indica LN2 and shows nematicidal activity against Heterorhabditis bacteriophora H06 nematodes.

Differentially expressed genes of Coptotermes formosanus (Isoptera: Rhinotermitidae) challenged by chemical insecticides.

Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) termites are harmful social insects to wood constructions. The current control methods heavily depend on the chemical insecticides with increasing resistance. Analysis of the differentially expressed genes mediated by chemical insecticides will contribute to the understanding of the termite resistance to chemicals and to the establishment of alternative control measures. In the present article, a full-length cDNA library was constructed from the termites induced by a mixture of commonly used insecticides (0.01% sulfluramid and 0.01% triflumuron) for 24 h, by using the RNA ligase-mediated Rapid Amplification cDNA End method. Fifty-eight differentially expressed clones were obtained by polymerase chain reaction and confirmed by dot-blot hybridization. Forty-six known sequences were obtained, which clustered into 33 unique sequences grouped in 6 contigs and 27 singlets. Sixty-seven percent (22) of the sequences had counterpart genes from other organisms, whereas 33% (11) were undescribed. A Gene Ontology analysis classified 33 unique sequences into different functional categories. In general, most of the differential expression genes were involved in binding and catalytic activity.

Genetic and proteomic characterization of rpoB mutations and their effect on nematicidal activity in Photorhabdus luminescens LN2.

Rifampin resistant (Rif(R)) mutants of the insect pathogenic bacterium Photorhabdus luminescens LN2 from entomopathogenic nematode Heterorhabditis indica LN2 were genetically and proteomically characterized. The Rif(R) mutants showed typical phase one characters of Photorhabdus bacteria, and insecticidal activity against Galleria mellonella larvae, but surprisingly influenced their nematicidal activity against axenic infective juveniles (IJs) of H. bacteriophora H06, an incompatible nematode host. 13 out of 34 Rif(R) mutants lost their nematicidal activity against H06 IJs but supported the reproduction of H06 nematodes. 7 nematicidal-producing and 7 non-nematicidal-producing Rif(R) mutants were respectively selected for rpoB sequence analysis. rpoB mutations were found in all 14 Rif(R) mutants. The rpoB (P564L) mutation was found in all 7 mutants which produced nematicidal activity against H06 nematodes, but not in the mutants which supported H06 nematode production. Allelic exchange assays confirmed that the Rif-resistance and the impact on nematicidal activity of LN2 bacteria were conferred by rpoB mutation(s). The non-nematicidal-producing Rif(R) mutant was unable to colonize in the intestines of H06 IJs, but able to colonize in the intestines of its indigenous LN2 IJs. Proteomic analysis revealed different protein expression between wild-type strain and Rif(R) mutants, or between nematicidal-producing and non nematicidal-producing mutants. At least 7 putative proteins including DsbA, HlpA, RhlE, RplC, NamB (a protein from T3SS), and 2 hypothetical proteins (similar to unknown protein YgdH and YggE of Escherichia coli respectively) were probably involved in the nematicidal activity of LN2 bacteria against H06 nematodes. This hypothesis was further confirmed by creating insertion-deletion mutants of three selected corresponding genes (the downregulated rhlE and namB, and upregulated dsbA). These results indicate that the rpoB mutations greatly influence the symbiotic association between the symbionts and their entomopathogenic nematode hosts.

Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite.

Serratia marcescens GEI strain was isolated from the gut of the workers of Chinese honey bee Apis cerana and evaluated in the laboratory for the control of Varroa destructor, a parasite of western honey bee A. mellifera. The supernatant and the collected proteins by ammonium sulfate from the bacterial cultures showed a strong miticidal effect on the female mites, with 100% mite mortality in 5days. Heat (100 degrees C for 10min) and proteinase K treatment of the collected proteins destroyed the miticidal activity. The improved miticial activity of this bacterial strain on chitin medium indicated the involvement of chitinases. The expressed chitinases ChiA, ChiB and ChiC1 from S. marcescens GEI by recombinant Escherichia coli showed pathogenicity against the mites in the laboratory. These chitinases were active in a broad pH range (5-9) and the optimum temperatures were between 60 and 75 degrees C. Synergistic effects of ChiA and ChiB on the miticidal activity against V. destructor were observed. The workers of both honey bee species were not sensitive to the spraying and feeding chitinases. These results provided alternative control strategies for Varroa mites, by formulating chitinase agents and by constructing transgenetic honey bees.

Identification and characterization of a novel gene involved in the trans-specific nematicidal activity of Photorhabdus luminescens LN2.

Photorhabdus luminescens subsp. akhurstii LN2 from Heterorhabditis indica LN2 showed nematicidal activity against axenic Heterorhabditis bacteriophora H06 infective juveniles (IJs). Transposon mutagenesis identified an LN2 mutant that supports the growth of H06 nematodes. Tn5 disrupted the namA gene, encoding a novel 364-residue protein and involving the nematicidal activity. The green fluorescent protein-labeled namA mutant was unable to colonize the intestines of H06 IJs.

Cloning and heterologous expression of insecticidal-protein-encoding genes from Photorhabdus luminescens TT01 in Enterobacter cloacae for termite control.

Enterobacter cloacae, one of the indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus), was genetically modified with a transposon Tn5 vector containing genes (tcdA1 and tcdB1) encoding orally insecticidal proteins from the entomopathogenic bacterium Photorhabdus luminescens subsp. laumondii TT01, a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora, for termite control. In the laboratory, termites were fed filter paper inoculated with the recombinant bacteria. The chromosomal expression of the introduced genes showed that there were insecticidal activities against termite workers and soldiers challenged with the transformed bacteria. After termites were fed recombinant bacteria, the termite mortality was 3.3% at day 5, and it increased from 8.7% at day 9 to 93.3% at day 29. All the dead termites contained the recombinant bacteria in their guts. Transfer of the recombinant bacteria occurred between donor workers (initially fed recombinant bacteria) and recipient workers (not fed). More than 20% of the recipient termites ingested recombinant bacteria within 2 h, and 73.3% of them had ingested recombinant bacteria after 12 h. The method described here provides a useful alternative for sustainable control of the Formosan subterranean termite (C. formosanus) and other social insects, such as the imported red fire ant (Solenopsis invicta).

[Effect of ethyl pyruvate on peroxidation injury of intestinal mucosa in rats with severe abdominal infection].

OBJECTIVE: To investigate the preventive effect of ethyl pyruvate (EP) on peroxidation injury to intestinal mucosa in rats with severe abdominal infection. METHODS: Thirty-six SD rats were divided randomly into three groups (n=30 in each group): control group (laparotomy only), infection group [cecal ligation and puncture (CLP) was performed to reproduce severe abdominal infection model] and EP group (CLP plus 40 mg/kg EP subcutaneous injection, once per 8 hours). The changes in intestinal mucosa pathologic score were observed, and malondialdehyde (MDA) and myeloperoxidase (MPO) activities in intestinal tissue, and serum MDA levels were determined at postoperative 24 and 48 hours. RESULTS: Inflammation of small intestine mucosa was more severe in the infection group than in EP group, and the pathologic scores were lower in EP group than those of the infection group at post-CLP 24 and 48 hours (all P<0.05). There was a significant positive correlation between the intestinal and plasma MDA in the infection group (r=0.867, P<0.05). The MDA and MPO levels in intestinal tissue and serum were higher in the infection group than in EP group and control group (all P<0.05). CONCLUSION: With severe intraperitoneal infection in rats, the intestinal mucosa is damaged by the reactive oxygen species. EP could ameliorate the injury of intestinal mucosa by attenuating the injurious effects of the reactive oxygen species.