The comparative analysis of Lonicerae Japonicae Flos and Lonicerae Flos: A systematical review.

ETHNOPHARMACOLOGICAL RELEVANCE: Lonicerae Japonicae Flos (LJF) and Lonicerae Flos (LF) were once used as the same herb in China, but they were distinguished by Chinese Pharmacopoeia in 2005 in terms of their medicinal history, plant morphology, medicinal properties and chemical constituents. However, their functions, flavor, and meridian tropism are the same according to the Chinese pharmacopoeia 2020 edition, making researchers and customers confused. AIM OF THE REVIEW: This review aimed to provide a comparative analysis of LJF and LF in order to provide a rational application in future research. MATERIALS AND METHODS: The information was gathered from China National Knowledge Infrastructure (CNKI), SciFinder, Google Scholar, PubMed, Web of Science, and Chinese Masters and Doctoral Dissertations (all chosen articles were reviewed attentively from 1980.1 to 2023.8). RESULTS: Till now, 507 chemical compounds have been isolated and identified in LJF, while 223 ones (79 overlapped compounds) are found in LF, including organic acids and derivatives, flavonoids, triterpenoids, iridoids, and essential oil components, etc. In addition, the pharmacological activities of LJF and LF, especially for their anti-influenza efficacy and mechanism, and their difference in terms of pharmacokinetic parameters, toxicology, and clinical applications were also summarized. CONCLUSION: The current work offers comparative information between LJF and LF in terms of botany, traditional uses, phytochemistry, ethnopharmacology, pharmacokinetics, toxicology, and pharmacology, especially their anti-influenza activities. Despite the same clinical applications and similar chemical components in LJF and LF, differentiated components were still existed, resulting in differentiated pharmacological activities and pharmacokinetics parameters. Moreover, the research about anti-influenza mechanism and functional substances of LJF and LF is dramatically limited, restricting their clinical applications. In addition, few studies have investigated the metabolism feature of LF in vivo, which is one of the important bases for revealing the pharmacological mechanism of LF. At the same time, the toxicity of LJF and LF is not fully studied, and the toxic compounds of LJF and LF need to be screened out in order to standardize the drug use and improve their rational applications.

Corrigendum: Effects of Xiao Chengqi formula on slow transit constipation by assessing gut microbiota and metabolomics analysis in vitro and in vivo.

[This corrects the article DOI: 10.3389/fphar.2022.864598.].

Growth of Enterococcus faecalis ∆plsX strains is restored by increased saturated fatty acid synthesis.

The Enterococcus faecalis acyl-acyl carrier protein (ACP) phosphate acyltransferase PlsX plays an important role in phospholipid synthesis and exogenous fatty acid incorporation. Loss of plsX almost completely blocks growth by decreasing de novo phospholipid synthesis, which leads to abnormally long-chain acyl chains in the cell membrane phospholipids. The ∆plsX strain failed to grow without supplementation with an appropriate exogenous fatty acid. Introduction of a ∆fabT mutation into the ∆plsX strain to increase fatty acid synthesis allowed very weak growth. The ∆plsX strain accumulated suppressor mutants. One of these encoded a truncated ß-ketoacyl-ACP synthase II (FabO) which restored normal growth and restored de novo phospholipid acyl chain synthesis by increasing saturated acyl-ACP synthesis. Saturated acyl-ACPs are cleaved by a thioesterase to provide free fatty acids for conversion to acyl-phosphates by the FakAB system. The acyl-phosphates are incorporated into position sn1 of the phospholipids by PlsY. We report the tesE gene encodes a thioesterase that can provide free fatty acids. However, we were unable to delete the chromosomal tesE gene to confirm that it is the responsible enzyme. TesE readily cleaves unsaturated acyl-ACPs, whereas saturated acyl-ACPs are cleaved much more slowly. Overexpression of an E. faecalis enoyl-ACP reductase either FabK or FabI which results in high levels of saturated fatty acid synthesis also restored the growth of the ∆plsX strain. The ∆plsX strain grew faster in the presence of palmitic acid than in the presence of oleic acid with improvement in phospholipid acyl chain synthesis. Positional analysis of the acyl chain distribution in the phospholipids showed that saturated acyl chains dominate the sn1-position indicating a preference for saturated fatty acids at this position. High-level production of saturated acyl-ACPs is required to offset the marked preference of the TesE thioesterase for unsaturated acyl-ACPs and allow the initiation of phospholipid synthesis.

Exploring the mechanism of Tengli Kangliu Decoction in the prevention and treatment of colorectal cancer precancerous based on network pharmacology.

OBJECTIVE: This study aimed to predict the targets and signaling pathways affected by Tengli Kangliu Decoction (TKD) in the treatment of colorectal cancer (CRC) precursor lesions and to determine TKDs mechanism of action based on previous experimental results using network pharmacology techniques and methods. METHODS: Using the traditional Chinese medicine systems pharmacology database (TCMSP) and UniProt database, the active ingredients and potential targets of TKD were identified. Human colorectal adenoma (CRA) targets were analyzed using the GeneCards database, the Online mendelian inheritance in man (OMIM) database, and the NCBI database. The common targets of drug-disease interactions were input into the String database to construct a protein-protein interaction (PPI) network. These data were then used to construct the network diagram. Gene ontology (GO) function analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed on the target genes. Finally, the component-disease-pathway-target network file was imported into Cytoscape 3.8.0 and used to construct the pathway network diagram. RESULTS: Compounds with a drug-likeness (DL) score ≥ 0.18 and an oral bioavailability (OB) ≥ 30% were selected as the active constituents of TKD. Two hundred eighty eight chemical constituents were screened and 305 chemical drug targets were predicted. After further screening, 1942 disease-related targets, which are hypothesized to be the main chemical components of TKD, were obtained. When comparing the targets of action and CRA treatment targets, 172 common targets were identified. Using GO enrichment analysis of common targets of drug diseases, 2550 biological processes (BP) were predicted, 164 items of which were related to molecular functioning (MF), and 67 items related to cell composition. KEGG pathway analysis was performed on the common targets of drug diseases, and a total of 178 signaling pathways were enriched. CONCLUSION: Using network pharmacology research, this study reports on the synergistic effect of the multiple components of TKD on the multi-target, and multiple pathways of colorectal precancerous lesions. These findings lay a theoretical foundation for further colorectal precancerous lesions research.

Phytochemicals, biological activity, and industrial application of lotus seedpod (Receptaculum Nelumbinis): A review.

Lotus (Nelumbo nucifera Gaertn.) is a well-known food and medicinal plant. Lotus seedpod (Receptaculum Nelumbinis) is the by-products during lotus products processing, which is considered as waste. Numerous studies have been conducted on its phytochemicals, biological activity and industrial application. However, the information on lotus seedpod is scattered and has been rarely summarized. In this review, summaries on preparation and identification of phytochemicals, the biological activities of extracts and phytochemicals, and applications of raw material, extracts and phytochemicals for lotus seedpod were made. Meanwhile, the future study trend was proposed. Recent evidence indicated that lotus seedpods extracts, obtained by non-organic and organic solvents, possessed several activities, which were influenced by extraction solvents and methods. Lotus seedpods were rich in phytochemicals categorized as different chemical groups, such as proanthocyanidins, oligomeric procyanidins, flavonoids, alkaloids, terpenoids, etc. These phytochemicals exhibited various bioactivities, including ameliorating cognitive impairment, antioxidation, antibacterial, anti-glycative, neuroprotection, anti-tyrosinase and other activities. Raw material, extracts and phytochemicals of lotus seedpods could be utilized as sources for biochar and biomass material, in food industry and as dye. This review gives well-understanding on lotus seedpod, and provides theoretical basis for its future research and application.

Electroacupuncture on Hemifacial Spasm and Temporomandibular Joint Pain Co-Morbidity: A Case Report.

Hemifacial spasm (HFS) and temporomandibular joint (TMJ) pain are common facial diseases which cause depression, anxiety, insomnia, and poor quality of life. However, currently there are still no effective therapies to treat HFS and TMJ. Electroacupuncture (EA) has advantages of safety, rapid work, easy operation and convenience. Here, we reported a case of a 50-year-old woman who presented with irregular spasm of eyelids and facial muscles on the left side, and TMJ pain on the right side. The patient had been treated with carbamazepine (20mg per day) and alternative therapies for a year, but still not much improvement in the symptoms. The scores of the Jankovic Rating Scale (JRS), global rating scale (GRS), and visual analog scale (VAS) were 7, 60, and 7 points, respectively. The EMG test showed that the spastic side had higher R1 amplitude, longer R2 duration, and larger R2 area than the non-spasmodic side, and the occurrence rate of the lateral spread responses (LSR) in the Orbicularis oris and the Orbicularis oculi muscle was 60% and 40%, respectively. We considered this patient had left HFS and right TMJ pain. EA was successfully undertaken for two periods over 30 weeks. After EA, JRS and VAS were reduced sharply, and the symptoms of HFS were stable without recurrence. However, the frequency of the lower eyelid increased gradually during the 6-month follow-up. These findings reveal that EA with the frequency of 2 Hz and intensity of ~ 1-2 mA may be a benefit for alleviating symptoms of HFS and TMJ pain without adverse reaction. The potential mechanisms of EA in HFS and TMJ pain co-morbidity involve brain stem mechanism and DNIC mechanism for distal acupuncture and segmental mechanism for local acupuncture analgesia.

Effects of Xiao Chengqi Formula on Slow Transit Constipation by Assessing Gut Microbiota and Metabolomics Analysis in vitro and in vivo.

The Xiao Chengqi (XCQ) formula is a newly constituted traditional Chinese medicine prescription in the treatment of intestinal motility deficiency and is effective in patients with slow transit constipation (STC). XCQ formula was reconstructed based on a "Chengqi" decoction. Astragali Radix, Angelicae Sinensis Radix, and cooked ground Salviae Miltiorrhizae Radix et Rhizoma were added to the prescription to enhance. An STC rat model was constructed and treated with the formula to understand the detailed mechanism by which XCQ promotes intestinal peristalsis. The effects of the XCQ formula on intestinal microflora and metabolic levels and the possible molecular mechanism of its regulation were explored using 16S rDNA sequencing, metabolomics sequencing, and tissue RNA sequencing. The results showed a significant decrease in the abundance of Roseburia spp. in the feces of STC rats, a significant decrease in the content of butyl aminobenzene (BAB) in feces, and an increase in the number of interstitial cells of Cajal (ICC) in the colon of STC rats. Furthermore, in vitro and in vivo experiments revealed that BAB could activate IL-21R on the ICC surface, upregulate the phosphorylation of the downstream molecules STAT3 and ERK, and inhibit loperamide-induced ICC apoptosis. Therefore, the XCQ formula can improve the defecation status of patients with STC by protecting ICC activity, promoting the colonization of Roseburia spp. to promote peristalsis, and increasing the BAB content after metabolism.

Xiaoyao Powder in the treatment of non-alcoholic fatty liver disease: A systematic review and meta-analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease worldwide with alarming prevalence. Due to its complex pathogeneses and considerable individual heterogeneity in disease, there is no specific medication to NAFLD safely and effectively. Therefore, there is a great need to explore complementary and alternative therapies. Xiaoyao Powder (XYP), a classic Chinese formula, has been tremendously applied to gastrointestinal diseases, especially non-alcoholic fatty liver disease. However, the efficacy and safety of XYP have not been fully assessed. AIM OF THE STUDY: To assess the effectiveness and safety of XYP for NAFLD. MATERIALS AND METHODS: The assigned registration number on the PROSPERO platform of this meta-analysis is CRD42020192154, and we strictly followed the protocol. We searched eight primary databases from their inception to June 2020. Two authors independently identified random controlled trials (RCTs) of XYP for NAFLD and evaluated the quality of the retrieved articles by Cochrane accessing risk bias tool. At least one of the following indices was thoroughly documented for outcome measurement: total effective rate, total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamyl transpeptidase (GGT), body mass index (BMI), and adiponectin. We calculated risk ratio (RR) and mean difference (MD) for dichotomous data and continuous variables with a 95% confidence interval (CI). R 4.0.5 software was employed for data synthesis. RESULTS: Consequently, we identified 12 studies with 1012 participants. XYP, whether individually or combined with essential treatment, ameliorated NAFLD regardless of the course of the disease or curative duration. This benefit was mainly driven by regulating levels of serum markers, involving TC, TG, ALT, AST, GGT, and adiponectin. Three studies where statins were concerned about drug safety reported several adverse events with clinical symptoms, varying from flatulence, constipation, and diarrhea to rash, whereas others did not. CONCLUSION: Our findings provided evidence that XYP is a therapeutic option to treat NAFLD effectively and safely. Notwithstanding, a precise and comprehensive conclusion calls for RCTs on a larger scale with more rigorous designs considering the inferior methodological quality and limited retrieved articles.

[Protective effect and mechanism of Angong Niuhuang pill in sepsis-associated brain dysfunction of rats].

OBJECTIVE: To observe the protective effect of Angong Niuhuang pill on brain function of rats with sepsis, explore its protective mechanism, and provide the experimental basis for clinical application of Angong Niuhuang pill in the treatment of sepsis-associated encephalopathy (SAE). METHODS: Thirty male Sprague-Dawley (SD) rats were divided into sham operation group, sepsis model group and Angong Niuhuang pill group according to random number table method, with 10 rats in each group. The sepsis model was established by cecal ligation and puncture (CLP); rats in sham operation group received open and closed abdomen. The rats in the Angong Niuhuang pill group were given Angong Niuhuang pill (0.3 g/kg) by gastric irrigation daily for 3 days before CLP, and the drugs were administrated 12 hours after modeling again. After 24 hours of CLP, the neuroreflex scores were evaluated, white blood cell count (WBC), the levels of serum neuron-specific enolase (NSE) and S100ß were detected. Then the brain tissue was harvested. After hematoxylin-eosin (HE) staining, the pathological changes of brain tissue were observed under the light microscope. The mRNA expressions of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in brain tissue were detected by polymerase chain reaction. RESULTS: Compared with the sham operation group, the total score of neuroreflex scores in the sepsis model group and the Angong Niuhuang pill group were significantly reduced (4.43±1.40, 6.57±1.90 vs. 9.40±0.84, both P < 0.05), WBC, serum NSE, S100ß were significantly increased [WBC (×109/L): 8.07±1.32, 5.84±0.94 vs. 3.60±0.32; NSE (µg/L): 1.04±0.14, 0.61±0.07 vs. 0.16±0.04; S100ß (ng/L): 255.624±30.25, 97.72±15.41 vs. 46.88±12.03, all P < 0.05], and the mRNA expressions of IL-6 and TNF-α in brain tissue were significantly increased [IL-6 mRNA (2-ΔΔCt): 5.668±2.195, 3.605±1.014 vs. 0.997±0.329; TNF-α mRNA (2-ΔΔCt): 18.996±0.913, 1.746±0.710 vs. 0.674±0.132, all P < 0.05]. Compared with the sepsis model group, the total score of neuroreflex scores in the Angong Niuhuang pill group was significantly increased (6.57±1.90 vs. 4.43±1.40, P < 0.05), WBC, serum NSE, S100ß concentration, and the mRNA expressions of IL-6 and TNF-α in the brain were significantly reduced [WBC (×109/L): 5.84±0.94 vs. 8.07±1.32, NSE (µg/L): 0.61±0.07 vs. 1.04±0.14, S100ß (ng/L): 97.72±15.41 vs. 255.62±30.25, IL-6 mRNA (2-ΔΔCt): 3.605±1.014 vs. 5.668±2.195, TNF-α mRNA (2-ΔΔCt): 1.746±0.710 vs. 18.996±0.913, all P < 0.05]. Brain histopathological observation showed that the hippocampal neurons in the sepsis model group were disordered arrangement, a large number of neuronal nuclei were contracted, and the tissue was loose with obvious edema. Compared with the sepsis model group, the Angong Niuhuang pill group had less nuclear shrinkage and tissue edema. CONCLUSIONS: The pretreatment of the Angong Niuhuang pill can improve the brain dysfunction of septic rats and reduce the expression of pro-inflammatory cytokines in the brain. It is speculated that the Angong Niuhuang pill can protect the brain function in sepsis by inhibiting the inflammatory reaction in the brain.

Antioxidant and hepatoprotective effects against acute CCl4 -induced liver damage in mice from red-fleshed apple flesh flavonoid extract.

Apple (Malus domestica Borkh.) is an important fruit tree species worldwide. Apple fruits are favored by consumers because of their antioxidative, anti-inflammatory, and antitumor effects as well as their protective effects against cardiovascular diseases and other health benefits. There is considerable interest in red-fleshed apple fruits among breeders because of their high flavonoid and anthocyanin contents. However, the flavonoids extracted from red-fleshed apple fruits must still be functionally characterized, especially regarding their protective effects against certain pathologies. In this study, the flavonoid components and contents in the extracts prepared from red-fleshed apple cultivar "Meihong" were determined. Additionally, the in vitro antioxidant activities and protective effects of the extracts against CCl4 -induced acute liver injury were investigated. The red-fleshed apple flesh flavonoid extract (RAFF) exhibited strong in vitro antioxidant activities. Compared with the model control mice treated with CCl4 , the mice pretreated with high (800 mg/kg·bw), middle (400 mg/kg·bw), and low (200 mg/kg·bw) RAFF doses had significantly lower CCl4 -induced serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities. Moreover, the RAFF pretreatment also significantly decreased the liver malondialdehyde activity and prevented the CCl4 -induced decrease in liver superoxide dismutase, glutathione peroxidase, catalase, and reduced glutathione levels. Furthermore, a histopathological examination revealed that RAFF inhibited the inflammatory cell infiltration and cell boundary loss caused by CCl4 in the liver. Thus, RAFF is a natural antioxidant with significant antioxidative activities and liver protective effects. The results of this study may be relevant for enhancing the application of the red-fleshed apple fruit extract as a food additive. PRACTICAL APPLICATION: We took the self-selected red-fleshed apple cultivar "Meihong" as the unique research material, and the active ingredients of its flavonoid extract, in vitro antioxidant activity and hepatoprotective effect were analyzed. It is of great significance to promote the development of the red-fleshed apple industry, and also provides an important reference for the development of natural antioxidants.

Administration of dietary recombinant hepcidin on grass carp (Ctenopharyngodon idella) against Flavobacterium columnare infection under cage aquaculture conditions.

Hepcidin links iron metabolism with innate immunity during the inhibition of bacterial infection. Our previous studies had shown that recombinant hepcidin can significantly reduce the mortality rate of Ctenopharyngodon idella infected with Flavobacterium columnare under laboratory conditions. Here, we studied the preventive and therapeutic effects of feed supplemented with different doses of recombinant hepcidin on F. columnare-challenged C. idella reared in a cage culture environment. The results showed that in the prevention groups, 30 and 90 mg/kg of added purified and unpurified hepcidin respectively resulted in a higher survival rate in the early post-infection period, while 60 mg/kg of purified hepcidin significantly improved the survival rate in the therapy group (all compared to the control group). In the hepatopancreas, the expression of hepcidin and ferritin was significantly up-regulated, and the levels of ferroportin and serum iron were significantly decreased, especially in the therapy group. In addition, the expression of iron-related genes in spleen and intestine exhibited a similar trend to that in hepatopancreas. Meanwhile, immune genes were up-regulated to varying degrees, and the therapy group exhibited a significantly improved expression of pro-inflammatory cytokines and specific immunity. In summary, our study shows that different doses of recombinant hepcidin had protective effects against bacterial infection by regulating the iron distribution and immune gene expression, which provides a strong foundation for the application of recombinant hepcidin in aquaculture.

[HPLC fingerprint of Zishen Yutai pills and simultaneous determination of 5 index components].

To establish the HPLC fingerprint and determine five index components (loganic acid， chlorogenic acid， loganin， sweroside and asperosaponin Ⅵ) of Zishen Yutai pills by high performance liquid chromatography, and provide a scientific basis for its quality control. The fingerprint chromatogram was analysed by the chromatographic fingerprint similarity evaluation system for tradition Chinese medicine (2012), fifteen common peaks were obtained at the wavelength of 254 nm. Different batches of Zishen Yutai pills showed a similarity of above 0.90 in HPLC fingerprint profiles. For the quantitive analysis method, The separation of five components showed good regression (r>0.999 2) with linear ranges, and the mean recoveries were in the range of 97.62%-101.9%, with the RSD (n=9) less than 3%. The established fingerprint and quantitative analysis methods are highly specific, simple and accurate, which can reflect the quality of Zishen Yutai pills more comprehensively, and can be used for its quality control.

Effects of binding materials on microaggregate size distribution in bauxite residues.

It is recognized that for successful establishment of a vegetation cover on bauxite residue disposal areas, soil formation and a greater understanding of the processes of soil development are crucial. The stability of microaggregates is a very important physical property that prevents erosion in bauxite residues. Samples were collected from a disposal area in Central China to determine not only the mechanism of aggregation but also clay dispersion. Colloidal stability was assessed by determining organic matter, carbonate, electrolyte, clay mineral, and iron-aluminum oxide forms, as these would contribute to their stability. Organic matter improved microaggregate stability by combining with clay particles and polyvalent cations to form macroaggregates. Polyvalent cations such as calcium had a positive effect on particle flocculation, while organic molecules were more effective at stabilizing microaggregates. Removal of salinity dispersed silt-size aggregates into clay-size aggregates and reduced microaggregate stability. Calcium improved particle aggregation, while sodium had the reverse effect. Quartz powder was added to the residues but did not show any cementing effect, while free and amorphous iron-aluminum oxides were effective binding agents for microaggregate formation. We propose that the presence of organic matter and polyvalent cations, together with incorporation of organic carbon and calcium minerals, may enhance the stability of this material and prove beneficial toward improving its physical condition.

[The effect of Xuebijing on liver translationally controlled tumor protein expression in Acinetobacter baumannii sepsis rats].

OBJECTIVE: To study the effect of Xuebijing on liver expressing translationally controlled tumor protein (TCTP) in Acinetobacter baumannii sepsis rats. METHODS: Among 42 healthy adult male Wistar rats of clean grade, 6 rats were randomly selected as the control group, others were randomly divided into two groups by the method of random digits table: sepsis group(n=18), Xuebijing group(n=18). Sepsis model was established through intraperitoneal injecting Acinetobacter baumannii suspension, and the Xuebijing injection was administrated through caudal vein 30 minutes later in Xuebijing group. After making model for 6, 12 and 24 hours, 6 rats were randomly selected from sepsis group and Xuebijing group, and then the rats were sacrificed, liver tissue samples were extracted for hematoxylin and eosin (HE) staining. Pathological changes of the liver were observed, and immunohistochemical analysis of liver tissue TCTP expression positive cells and the expression of TCTP in liver cells were detected by Western blotting method. RESULTS: HE staining of liver indicated that it was inflammatory injured in sepsis group, and inflammation decreased in Xuebijing group. Immunohistochemistry results showed that, compared with the control group, TCTP positive cells expression score at 6, 12 and 24 hours in sepsis group were significantly increased (7.33±0.82, 10.67±1.21, 7.67±1.21 vs. 2.50±1.05, all P<0.05). Compared with sepsis group, liver tissue TCTP positive cells expression score at 6, 12 and 24 hours in Xuebijing group (5.83±0.75, 7.50±1.05, 5.67±1.37) were significantly decreased (all P<0.05). Western blotting results showed that, compared with the control group, TCTP expression at 6, 12 and 24 hours in sepsis group were significantly increased (1.94±0.59, 3.20±0.72, 1.96±0.55 vs. 0.93±0.24, all P<0.05); compared with sepsis group, TCTP expression at 6, 12 and 24 hours in Xuebijing group (1.38±0.36, 2.03±0.49, 1.30±0.30) were significantly decreased (all P<0.05). CONCLUSIONS: Xuebijing can reduce inflammatory injury in liver of rats with Acinetobacter baumannii sepsis, and its mechanism may be associated with reduced hepatic cells expressed TCTP.

Gene cloning and expression patterns of two prophenoloxidases from Catantops pinguis (Orthoptera: Catantopidae).

In insect, fat body plays major roles in insect innate immunity. Phenoloxidase (PO) is an important component in insect innate immunity and is necessary for acclimatization. In our study, two prophenoloxidase (PPO) subunits were obtained from fat body of Catantops pinguis (Stål). The full-length cDNA sequence of one PPO (CpPPO1) consisted of 2347 bp with an open reading frame (ORF) of 2187 bp encoding 728 amino acids, while the other subunit (CpPPO2) had a full length of 2445 bp, encoding 691 amino acids. Both the PPO gene products are predicted to possess all the structural features of other PPO members, including two putative tyrosinase copper-binding motifs with six highly conserved histidine residues and a thiolester-like motif. Tissue distribution analysis showed that both PPO mRNAs were abundantly expressed in the fat body among 11 tissues examined, and they were transiently up-regulated after Escherichia coli infection, consistent with them being immune-responsive genes. Total levels of CpPPO1 and CpPPO2 mRNA transcripts were much higher in first instar larvae and adults. A much higher transcript level of CpPPO1 was detected in several months, while there were extremely high mRNA expression levels of CpPPO2 in January, July, October, and December. The above results suggested that PPO from fat body might also bring significant function during the processes of development and acclimatization for C. pinguis.