Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 4934890, 11 pages https://doi.org/10.1155/2018/4934890

Review Article Pharmacological Properties of the Medical Maggot: A Novel Therapy Overview

Litao Yan ,1 Jin Chu,1 Mingshu Li,2 Xianfeng Wang,1 Junwei Zong,1 Xueyang Zhang,3 Mingzhi Song ,1,4 and Shouyu Wang 1

1 Department of Orthopaedics, Te First Afliated Hospital of Dalian Medical University, Dalian, Liaoning, China 2Department of Ophthalmology, Te First Afliated Hospital of Dalian Medical University, Dalian, Liaoning, China 3Department of Endocrinology, Te First Afliated Hospital of Dalian Medical University, Dalian, Liaoning, China 4Department of Orthopaedics, Te Tird Afliated Hospital of Dalian Medical University, Jinpu New District, Liaoning, China

Correspondence should be addressed to Mingzhi Song; [email protected] and Shouyu Wang; [email protected]

Received 14 November 2017; Accepted 5 April 2018; Published 3 May 2018

Academic Editor: Ronald Sherman

Copyright © 2018 Litao Yan et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In the last decade, maggot has been hailed as the miraculous “medicinal maggot” for its diverse properties, including antimicrobial, antibioflm, anti-infammatory, and wound healing activities. Te fact that maggots show so many benefcial properties has increased the interest in these tiny larvae dramatically. Whilst there is relatively abundant clinical evidence to demonstrate the success of maggots as debridement agents, not so much emphasis has been placed on the basic science evidence, which was a combination of physical and biochemical actions. Tis review difers from those earlier works in that it is undertaken to provide an update of the latest scientifc basis published on maggot, particularly active ingredients within maggot excretions/secretions (ES). Further investigations should focus on the isolation, identifcation, recombination, transgenosis, and mass production of the benefcial molecules within maggots.

1. Introduction to be benefcial in clinic and then studied in the laboratory. Finally,thespeciesisfurtherusedformorepreciseclinical MaggotisthelarvaeofLucilia sericata,belongingtothe applications. It is shown promising for therapeutic applica- family Calliphoridae within the order Diptera, which is one of tion with less expense and adverse efect, compared with the traditional Chinese herbal medicines. In ancient China, it conventional therapeutic methods. Tough there is a growing was described as “quality of cold, favour, invigorating spleen, acceptance of clinical applications of the maggot, along with heat to eliminate rickets” in the Compendium of Materia extensive clinical trials to demonstrate the success of MDT, Medica and was widely used in folk medicine to treat infantile scientifc laboratory evidences are still not enough to reveal malnutrition. Worldwide, live maggot has been used to clean wounds the biochemical mechanisms underlying the benefcial efects by degrading, liquefying, and ingesting only devitalized/ of the maggot. At present, the hot research mainly focuses necrotic tissues for centuries. Tis biotherapy is well known ontheantibacterialefects.Temaggotactuallyhasother as the maggot debridement therapy (MDT) [1]. Zacharias diverse natural properties, such as favorable efects on anti- andJoneswerethefrstthatappliedmaggotstothewounds infammatory, angiogenesis, extracellular matrix remodelling during the American Civil War. However, it was not until as well as fbroblast migration/proliferation. 2004 that the permission was granted for the use of medical Here, we collect the latest basic research data published maggots by Food and Drug Administration (FDA) [2]. on maggot in recent 20 years, particularly active ingredients Te MDT has experienced a cycle of “clinics--laboratory of maggot ES, and provide convincing molecular evidence to evidences--clinics”. Put simply, the maggot was frst found prove the decisive role of maggots in biotherapy. 2 Evidence-Based Complementary and Alternative Medicine

2. Antibacterial Activity with antibacterial activity against Micrococcus luteus and P. aeruginosa were also isolated from the maggot, including p- Wound clearing is the most important stage of MDT, hydroxyphenylacetic acid (152 Da), p-hydroxybenzoic acid which means removing necrotic and contaminated tissues. In (138 Da), and proline diketopiperazine (194 Da). Moreover, response to bacterial challenges, maggot was found to ingest theefectwasevenmoreenhancedwhenthesemolecules and kill E. coli when it passed through the midgut using were tested in combination [14]. confocal microscopy [3]. Tere was still another suggestion of In the last decade, a defensin peptide, lucifensin, from antibacterial mechanism which was called simple mechanical the ES and body tissues (fat body, haemolymph, and gut), irrigation by ingesting liquefed necrotic tissues [4]. was frstly reported. It consisted of 40 amino acid residues Long before in 1935, Simmons had revealed the presence and three intramolecular disulphide bridges. Te authors of an efective bactericide in maggot ES, which exhibited a surmised that lucifensin was that long-sought antimicrobial potent and rapid disinfection action. Te maggot ES con- factor of the maggot ES [15]. Andersen et al. used molec- sisted of salivary gland secretions and faecal waste products ular biology methods to detect the sequence of lucifensin [5, 6]. and found it was active against a range of Staphylococ- Numerous investigations have since focused on antibac- cus and Streptococcus species. As a homologue to other terial activity in maggot ES against both Gram-positive and Gram-negative bacteria. However, there were many dipteran defensins, lucifensin had a similar antibacterial contrary conclusions. For example, Tomas et al. confrmed activity against Gram-positive bacteria [16]. Te expression of the antibacterial activity of secretions against a range of lucifensin was strongly stimulated in the fat body when it was bacteria, including Streptococcus A and B, Staphylococcus exposed to S. aureus and P. ae r ug ino s a in vitro. However, the aureus, Pseudomonas, and even methicillin resistant S. aureus antimicrobialactivityofESwasnotenhancedbypathogens (MRSA) in a preliminary laboratory study. No evidence of from the environment [17]. In the same year, lucifensin inhibition was shown against neither Enterococcus nor E. II, diferent from that of lucifensin by only one amino coli or Proteus [7]. But aferwards, Barnes et al. provided a acid residue, was found from hemolymph of the maggot contradictoryresultthatmaggotESwasmorepotentagainst [18]. As a kind of cationic antibacterial peptides, lucifensin E. coli than both S. aureus and P. ae r ug ino s a . Tey believed andlucifensinIIwerebelievedtokillbacteriabyforming that multiple factors such as the number of maggots, bacterial ion channels or transmembrane pores, causing leakage of species, and type of assay could infuence its antibacterial cytoplasmic components from bacterial cell membrane [18, potency and thus a standard method to quantify antibacterial 19]. In a pioneering work, lucilin, a 36-residue cecropin- activity should be established to make the result consistent like antimicrobial peptide, was identifed as a partial genetic and comparable [8]. Interestingly, it was observed in clinic sequence in maggots [20]. GWLK-Lucilin-CPD-His8, the thatMDTwasamoreefectivestrategytomanageGram- form of recombinant fusion protein with a cysteine pro- positive bacteria (e.g., S. aureus)thanGram-negativebacteria tease domain, was active against multidrug resistance Gram- (e.g., P. ae r ug ino s a ). Te authors indicated that a sufcient negative bacteria and without hemolytic activity towards number of maggots were needed not only for a larger wound, human erythrocytes. but also to exert an activity against Gram-negative bacteria Zhang et al. isolated and purifed an antibacterial pro- [9]. tein (<10 kDa) from maggots, named antibacterial protein Long time ago, it was shown that the maggot ES contained from maggots (MAMP), which demonstrated inhibitory kinds of alkaline compounds (e.g., ammonium carbonate, activity against both standard strains and clinically isolated calcium, allantoin, and urea) that inhibited bacterial growth antibiotic-resistant strains of S. aureus.Troughscanning byrisingpHinwounds,aswellasprovidingtheoptimum electron and transmission electron microscopy, the authors environment for proteolytic enzyme activity. Over the past supposed the possible mechanism was the interaction with two decades, many researchers have focused on identifying the bacterial cell membrane and disruption of the cell surface and isolating antimicrobial molecules produced by maggots. structure, leading to the increase of membrane permeability Such investigations are driven partly by the dilemma of [21]. microorganisms resistance (e.g., MRSA) [10]. On a further experiment, Bexfeld et al. discovered that maggot ES exhibited potent and antibacterial activity against 3. Antibiofilm Activity MRSA in vitro. Two molecular mass fractions (<500 Dalton (Da) and 0.5–10 kDa) with thermal stability and protease Maggots also fght bacteria in their more resistant form: resistance were partially isolated using ultrafltration tech- bioflm, which is a structured community living closely in a niques [11]. Te authors carried out an exhaustive study protective and self-produced polymeric matrix. It is widely on the antibacterial activity of the <500 Da fraction against accepted that bioflm associated infections are notoriously a range of bacteria. Distinct morphological changes were difcult to treat, even with advanced antibiotics. observed in Bacillus cereus and E. coli [12]. Te <500 Da Morerecently,astudydemonstratedthatmaggotESwere fraction was accurately determined as the empirical formula diferentially efective against bioflms of S. aureus and p. C10H16N6O9 and the molecule was patented and registered as aeruginosa.ItwasfoundthatEScoulddisruptbioflmfor- a novel antibiotic, Seraticin [13]. On the basis of above exper- mation of S. aureus at a very low concentration of 0.2 �gand iment, three categories of low molecular weight compounds rapidly degrade bioflms with the concentration increasing Evidence-Based Complementary and Alternative Medicine 3 to 2 �g. Te collapse of p. aeruginosa bioflm required a high 4. Synergistic Effect with Antibiotics concentration of 20 �g[22]. ButwhatmakesmoresenseisthatmaggotESmayact Clinically, a combination of maggot ES with existing medical selectively against diferent bacterial strain. In the study of treatments may possess greater therapeutic potential. Tere- Bohova et al., ES were efective in the reduction of bioflm fore, van der Plas et al. performed a study to assess the efect formation and the eradication of established bioflms of of combination of maggot ES and antibiotics (vancomycin, S. aureus and E. cloacae.Onthecontrary,ESstimulated daptomycin, and clindamycin) on S. aureus bioflms. It was P. mirabi li s bioflm formation surprisingly [23]. Tey also suggested that the combination of maggot ES and antibiotics foundmaggotESeradicatethebacterialbioflmofdiferent could break down S. aureus bioflms thus allowing the bacterial strains through diferent mechanisms. In the case bacterial cells exposure to the antibiotics. Tey indicated that of E. cloacae, preformed bioflm and viable cells were all responsible molecules in maggot ES were proteases belonging disrupted. However, ES only prevented bioflm formation in to the group of serine proteases [31]. terms of S. aureus. Tis was consistent with the previous Ten, a similar study was investigated whether maggot ES observations [7, 24]. infuence the antibacterial activity of diferent antimicrobial agents. Te result showed a dose-dependent increase of the Cazander et al. found that maggot ES prevent and inhibit antibacterial efect of gentamicin and fucloxacillin in the P. ae r ug ino s a bioflm formation. Te bioactivity of ES was presence of ES on S. aureus [32].TecombinationefectofES still stable for 1 month at room temperature [25]. Moreover, and ciprofoxacin also showed enhanced antibacterial activity in another study [26], the authors investigated the bioflm against S. aureus at subinhibitory concentration [33]. formation of other bacterial species (S.aureus,S.epidermidis, K. oxytoca, E. faecalis,andE. cloacae) on several biomaterials (polyethylene, titanium, and stainless steel). Te observations 5. Antifungal Bioactivity suggested maggot ES could decrease bioflm formation and So many studies have sought to determine the antibacterial provide an innovative treatment for bioflm formation on components present in maggot ES. However, less well known infected biomaterials. aretheliteratureswithregardtotheantifungalproperties. Te specifc mechanism was unclear; therefore, Harris et As recorded by Alnaimat et al., the maggot ES contained al. explored the interaction of ES and bioflm formation using a lot of alkaline compounds (e.g., ammonium carbonate, two strains of S. epidermidis (1457 and 5179-R1). Tey found allantoin),whichshowedmoderateantifungalactivityagainst that mechanisms involved in bioflm formation were through yeast and mould [34]. Poppel¨ et al. reported a novel antifungal the degradation of the polysaccharide intercellular adhesin peptide from the maggot, named lucimycin. Te molecular (PIA) and accumulation associated protein (AAP), respec- weight of this peptide was 8.2 kDa with 77 amino acid tively. Inhibitory activity was sensitive to heat treatment, > residues [35]. Lucimycin was active against fungi from the andtheparticularresponsiblemoleculewasinthe 10 kDa Ascomycota, Basidiomycota,andZygomycota,tothePhytoph- fraction, appearing to have protease or glucosaminidase thora parasitica, but it was inactive against bacteria. Te activity [24]. authors suggested that the lucimycin was likely to bind zinc In another investigation, chymotrypsin 1 derived from and probably other divalent metal ions. Tus the mechanism maggot ES was found to be responsible for digesting colla- was involved with not only the overall physicochemical gens, which could be conducive to impede bacterial colonisa- properties of the peptide but also the metal complex for- tion and subsequent bioflm formation [27]. Afer that, Harris mation to a specifc receptor. In 2015, a paper published by et al. became interested in the recombinant chymotrypsin Evans et al. demonstrated that maggot ES were active against (rChymotrypsin) isolated from maggot ES. Tey studied Candida albicans andappearedtobehighlyheatstable the potential of this rChymotrypsin when it interfered with and lyophilization resistant. Te antifungal components were staphylococcal bioflms in the way similar to the above isolated in three fraction masses, >10, 10–0.5, and <0.5 kDa. experiment. Compared with S. epidermidis 1457 and S. aureus Te strongest level of activity was seen in the <0.5 kDa SA113, the greatest efect of rChymotrypsin on both bioflms fraction [36]. was seen on S. epidermidis 5179-R1, which has suggested that Because of the lack of further experiments, the mech- maggot rChymotrypsin disrupted protein adhesin-mediated anism of antifungal activity is still unclear, but it probably bioflm formation of Staphylococcus [28]. difers from the antimicrobial mechanism of classical cationic Brown et al. purifed a native molecular mass from the AMPs. Hence, more eforts should be put to identify and maggot ES, which was demonstrated to have a deoxyri- chemically defne the antifungal components. Meanwhile, a bonuclease (DNAse) activity. Tis maggot DNAse, approxi- greater range of fungal pathogens and minimum inhibitory mately 45 kDa with magnesium, sodium, and calcium metal concentration testing should be carried out. ion dependency, degraded extracellular bacterial DNA in bioflms preformed from P. ae r ug ino s a . In addition, the 6. Anti-Inflammatory Activity DNAse was able to digest the DNA found in slough/eschar [29]. From an in vitro experiment, Cowan et al. demonstrated Infammation is the frst stage of wound healing. How- that maggot was efective in eliminating mature S. aureus ever, excessive infammatory reaction is blamed for chronic bioflm from experimental pigskin explants within 24 hours wounds. Neutrophils, monocytes, and macrophages are main [30]. producers of ROS along with kinds of proinfammatory 4 Evidence-Based Complementary and Alternative Medicine cytokines, therefore preventing the wound moving forward were upregulated in the presence of the BLIP. It was believed healing process. that the BLIP acted as an important immune evasion role Te maggot ES was discovered to signifcantly reduce to inhibit T lymphocyte activation when encountering a the level of both superoxide generation and myeloperoxidase stimulus [43]. Zhang et al. also found that the crude extracts (MPO) released from stimulated neutrophils rather than from maggots could raise the level of serum hemolysin and unstimulated neutrophils [37]. Afer that, van der Plas et the data of carbon expurgatory test in a murine model [44]. al. assessed the efects of maggot ES on human neutrophils, monocytes, and macrophage in three associated experiments. 8. Proangiogenic Activity In the frst one [38], H2O2 production via fMLP- and PMA- activated neutrophils was found to be inhibited by maggot ES Healing of infammation ofen involves growth of granulation in a dose-dependent manner. Meanwhile, maggot ES reduced tissue, which is composed mainly of capillaries and fbrob- fMLP-stimulated expression of CD11b/CD18 and inhibited lasts. Both laboratory and clinical studies about maggot- neutrophil chemotaxis towards fMLP. Te authors suggested induced wound healing revealed profound angiogenesis [45]. that maggot ES inhibit the proinfammatory responses of Te research from Wang et al. supported the notion human neutrophils through a cyclic AMP-dependent mecha- that maggot ES promoted angiogenesis directly by inducing nism. To confrm the supposition, they carried out the second human microvascular endothelial cells (HMEC-1) migration experiment [39] and found that proinfammatory cytokines and this event was partially mediated by the activation (IL-12p40, TNF-�, and MIF) from lipopolysaccharides- of AKT1, but not ERK1/2. Tis wound healing assay also stimulated monocytes were decreased by maggot ES, whereas indicated that maggot ES improved cell migration activity the anti-infammatory cytokine IL-10 was enhanced. Finally, rather than increasing cell proliferation [46]. they drew the conclusion that maggot ES inhibited the proin- van der Plas et al. found that maggot ES enhanced the fammatory responses of human neutrophils and monocytes production of proangiogenic growth factors, such as vascular through elevation of cyclic AMP. Moreover, maggot ES also endothelial growth factor (VEGF) and basic fbroblast growth decreased the chemotactic response of monocytes to fMLP, factor (bFGF) by macrophages [40]. Aferwards, three amino which coincided with the earlier conclusion of reducing the acids (histidine, valinol, and 3-guanidinopropionic acid) migration of human neutrophils towards fMLP.Of note, mag- were identifed within maggot ES and specifcally exhibited gotESdidnotafectthephagocytosisandintracellularkilling signifcant proangiogenic efect on human umbilical vein of Staphylococcus aureus/Candida albicans by monocytes or endothelial cells (HUVECs), whilst having no efect on neutrophils [38, 39]. According to third experiment [40], it fbroblasts [47]. Te result was confrmed by Sun et al. was shown that monocyte-macrophage diferentiation was who found that maggot ES promoted HUVEC proliferation altered by maggot ES from a proinfammatory to a proangio- and increased expression of VEGF receptor 2. Moreover, genic type in the presence of maggot ES. Additionally, MØ-1 a signifcant elevated level of CD34 and CD68 was also and MØ-2 were regulated by maggot ES with an increased observed, which was indicative of promoting angiogenesis production of MCP-1 and IL-8 and a reduced production of [48]. As well, another component extracted from maggot, the chemokines MIP-1b, RANTES, and PDGF-BB. fatty acid, was found to be favor of stimulating angiogenesis In 2012, Cazander et al. frstly investigated the efect of in a murine wound model via increasing VEGF expression maggot ES on complement activation (CA) in sera obtained (VEGFA mRNA and VEGFA protein expression) and wound from patients preoperatively and postoperatively. Maggot ES capillary density. From the analysis of GC/MS, 80% of the clearly reduced CA via all pathways, underlying the possible extracts were unsaturated and probably were active ingredi- mechanism of breaking down complement proteins C3 and ents [49]. C4 in a cation-independent manner. Te responsible ES Honda et al. reported a signifcant increase in endogenous component was supposed to be thermostable and has not hepatocyte growth factor (HGF) from blood samples of been clearly identifed [41]. In a further study, it was con- patients treated with MDT. Te authors suggested that ES frmedthatmaggotESinhibitedcomplementactivationby promoted HGF production via a positive feedback loop of two diferent mechanisms and downregulated the C3a/C5a- HGF--c-MET--STAT3 and then promoted healthy granula- mediated neutrophil activation [42]. tion tissue [50]. Te STAT3 signaling pathway was confrmed by Li et al. in a recent study. Te authors detected fve � 7. Immunomodulatory Function signaling pathways (Wnt2-, NF- B-, Notch1-, STAT3-, and TGF-�/Smad3), which are involved in wound healing and In fact, immune modulation by maggot is a protective suggested that two signaling pathways (STAT3 and TGF- behavior in the feld of MDT. Elkington et al. characterized �/Smad3) were activated in the presence of maggot extracts, a 56 kDa protein, blowfy larval immunosuppressive protein accompanied with their downstream gene expression (c-Myc, (BLIP), from maggot ES and identifed it as a member of VEGF, and cyclin D1) [51]. the serpin protein family. Te authors found that maggot ES were capable of inhibiting mitogen-induced ovine T 9. Fibroblast Migration/Proliferation and lymphocyte proliferation by decreasing expression of the ECM Remodelling activation marker CD25. Subsequently, the expression of the cytokine genes (IFN-�, IL-4, IL-10, and IL-13) was down- All kinds of trauma could cause diferent degrees of degener- regulated. Contrarily, TNF-� and TGF-� gene expressions ation, necrosis, and tissue defect; therefore cell proliferation Evidence-Based Complementary and Alternative Medicine 5 and EMC remodelling are prerequisite for tissue repair. �-chymotrypsin was detected in another study [59]. Te Fibroblasts migrate from the edge of the wound and are recombinant maggot chymotrypsin I still remained active crucial to the formation of granulation for reconstructing within wound eschar; however, human �-chymotrypsin the wound defects. Fibroblasts also secrete proteases (e.g., was inhibited by endogenous �1-antichymotrypsin and �1- serine proteases and matrix metalloproteinases) which are antitrypsin. responsible for reorganization of the components within ECM. 10. Procoagulant Activity In 1997, Prete frstly demonstrated that maggot ES/hemo- lymph stimulated the proliferation of fbroblasts and did not Te infuence of maggot on blood coagulation has not been merely shif the kinetics of fbroblast growth. Furthermore, studied in detail so far. Until 2015, kahl et al. frstly identifed inthepresenceofepidermalgrowthfactor(EGF),maggot and characterized the procoagulant properties of maggot ES/hemolymph signifcantly caused additional fbroplasia, ES [60]. Te specifc (chymo-) trypsin-like serine proteases suggesting a diferent mechanism to that of EGF, or a syn- induced clotting of human plasma and whole blood, partic- ergistic efect [52]. Recently, Smith et al. found that maggot ularly by activating contact phase proteins factors XII, IX, ES markedly accelerated the migration of fbroblasts and andkininogen.Terewasnoobviousinfuenceonplatelet epidermal keratinocytes during wound closure without any activation or fbrinolysis. More recently, the authors separated signifcant mitogenic efect [53]. the maggot ES and analysed their components, resulting Te study by Horobin et al. confrmed the role of maggot in the identifcation of a chymotrypsin-like serine protease, ES proteinases, mainly serine proteinases, in promoting Jonah-like protein. Tis protein was shown to reduce the fbroblast migration. Te authors pointed out that maggot clotting time of human plasma, even in the absence of the ES promoted human dermal neonatal fbroblast (HDNF) endogenous protease kallikrein, factors XI, XII [61]. cell migration, which was correlated to the degradation of fbronectin upon a fbronectin-coated surface [54]. In order 11. Neuranagenesis to elucidate the mechanism behind the efect, the same authors developed a three-dimensional model to observe Zhang et al. found that the homogenate products from fbroblastmigrationandmorphologyinresponsetomaggot disinfected maggot increased protein gene product 9.5 (PGP ES.Tisnovelmodel,whichmorecloselymimickedthe 9.5) expression and substance P secretion in the murine microenvironment in vivo, provided a much better under- cutaneous wound model [62]. PGP 9.5 is localised in normal standing of the interactions between the ECM, resident neural and neuroendocrine tissues [68]. Substance P is cells,andmaggotESinthewoundhealingprocess.Possible mainly present in sensory nerve endings of skin tissue and has mechanisms may be through the promotion of fbroblast neurotransmitter and neuromodulatory efects [69]. Tus, motility, release of bioactive compounds from the ECM, the authors hypothesized that maggot homogenate products and coordination of cellular responses [55]. Polakovicovaˇ et could promote wound nerve regeneration and neuropeptides al.usedaninvitromodelsimilartothatofHorobinto release. Recently, Chu and Yan demonstrated that PGP observe the morphologic properties of fbroblasts exposed to 9.5 was highly expressed in wounds treated with maggots, maggotES.Teauthorsobservedthat,afer5and10days comparedwithmupirocin[63].However,therewasstillalack of cultivation with maggot ES, there was an increased cell of enough basic research in the feld of neuranagenesis. metabolism and protein production (secondary lysosomes and residual bodies) to produce the microfbrillar network 12. Antitumor Activity which was necessary for their migration and the remodelling of the ECM [56]. Te fatty acids (FA1 and FA2) extracted from maggot showed Chambers et al. made a systematic survey of the prote- remarkable inhibitory activities against human leukemia cells olytic activity of maggot ES against physiologically relevant HL-60andhumanlungcancercellsA-549invitro[64]. ECM components in vitro [57]. Tree classes of prote- Te main active component of the fatty acids was polyun- olytic enzyme were detected in the maggot ES, including saturated fatty acid (PUFA), especially �-6 PUFA. Zhang metalloproteinases, aspartyl proteases, and serine of two et al. established a H22 hepatoma-bearing mice model and diferent subclasses (trypsin-like and chymotrypsin-like). discovered that maggot ES could inhibit H22 tumor growth Chymotrypsin-like serine proteinases were predominant to possibly through the activation of p38 mitogen-activated solubilize fbrin clots and degrade ECM components, such protein kinase (p38MAPK) signal pathway [65]. as fbronectin, laminin, and acid-solubilized collagen types I and III. Te trypsin-like activity appeared to be crucial 13. Antiatherosclerosis Activity to PAR-mediated activation of proliferation or cytokine secretion within the wound. Following this investigation, a In an atherosclerosis mice model, maggot ES showed certain chymotrypsinogen from the maggot has been successfully antiatherosclerosis efect that could decrease the serum level cloned [58]. Compared with the chymotrypsins from bovine of triglyceride (TG), total cholesterol (TC), and low density and human sources, the active recombinant chymotrypsin lipoprotein (LDL) and increase the level of high density I was superior to degrade eschar ex vivo, which indicated lipoprotein (HDL) [66]. Moreover, it could efectively regu- a better proteolytic activity. Te inhibition profle of the late proliferation, diferentiation, and secretory function of recombinant chymotrypsin I in comparison with human lymphocytes along with CD4+/CD8+ ratio in atherosclerosis, 6 Evidence-Based Complementary and Alternative Medicine [15] [13] [18] [21] [14] [10] [39] [27] [38] [43] [29] [20] [40] [31–33] [41, 42] [35, 36] Reference � � , MIF e u l and TGF- a � , IL-4, IL-10, v � H - - - P pores e and C4 receptor s i PDGF-BB antibiotics Mechanism a IL-13, and CD25 Increase in IL-10 bioflm formation proangiogenic type concentration (MIC) Digest DNA in bioflm Increase MCP-1 and IL-8 Reduce minimal inhibitory bacterial cells exposure to the Inhibit neutrophil chemotaxis Inhibit monocytes chemotaxis Upregulate TNF- Reduce superoxide generation, Lysis of the bacterial membrane Break down bioflm to allow the Decrease� ,RANTES,and MIP-1 Increase membrane permeability Downregulate IFN- Disrupt protein adhesin-mediated Decrease in IL-12p40, TNF- myeloperoxidase, and CD11b/CD18 Cyclic AMP-dependent mechanism Break down complement proteins C3 Metal complex formation to a specifc Form ion channels or transmembrane Diferentiate from proinfammatory to , -R Object species aeruginosa Monocytes Staphylococcus aureus Neutrophils clindamycin Macrophage Ciprofoxacin T lymphocyte Complement system Multidrug resistance Staphylococcus aureus Staphylococcus aureus Phytophthora parasitica Gram-negative bacteria Pseudomonas aeruginosa Pseudomonas aeruginosa Gentamicin, fucloxacillin Vancomycin, daptomycin, Staphylococcus epidermidis Ascomycota, Basidiomycota, Staphylococcus Streptococcus Bacillus cereus Escherichia coli Zygomycota, Candida albicans, strains of Standard and antibiotic-resistant Micrococcus luteus, Pseudomonas Table 1: Overview of the bioactivities of maggot. ES ES ES BLIP Lucilin DNAse MAMP Seraticin Allantoin Lucifensin Lucimycin Lucifensin II Chymotrypsin 1 Serine proteases Ammonium carbonate p-Hydroxybenzoic acid Proline diketopiperazine p-Hydroxyphenylacetic acid Kind of thermostable protein Substance/component/molecule Bioactivity Antibacterial Antibioflm Synergistic efect with antibiotic Antifungal Anti-infammation Immunomodulatory Evidence-Based Complementary and Alternative Medicine 7 [61] [57] [47] [49] [50, 51] [54, 55] [62, 63] [64, 65] [66, 67] Reference � /Smad3 cyclin D1 Mechanism Increase HDL residual bodies) signaling pathway IX, and kininogen Improve migration Improve migration substance P) release Degrade fbronectin Reduce clotting time Solubilize fbrin clots Decrease TC, TG, LDL Digest ECM components Regulate CD4+/CD8+ ratio Downregulate c-Myc, VEGF, and Activate AKT1 signaling pathway Activate p38MAPK signal pathway Activate STAT3 and TGF- Activate contact proteins factors XII, Increase cell metabolism and protein Enhance VEGFR-2, CD34 and CD68 Promote neuropeptides (PGP 9.5 and production (secondary lysosomes and - Object Whole blood Murine serum H22 hepatoma endothelial cell Human plasma EMC components Human leukemia cells Human umbilical vein A-549 lung cancer cells Fibroblasts keratinocytes Hepatocyte growth factor Table 1: Continued. Microvascular endothelial cell ES acid serine Histidine � -6 PUFA Not identifed Jonah-like protein Aspartyl proteases Metalloproteinases Unsaturated fatty acid Homogenate products Kind of serine proteinases Trypsin/chymotrypsin-like Valinol 3-guanidinopropionic Substance/component/molecule Bioactivity Proangiogenic Fibroblast migration/proliferation and ECM remodelling Procoagulant Neuranagenesis Antitumor Antiatherosclerosis 8 Evidence-Based Complementary and Alternative Medicine which may be one of the mechanisms of antiatherosclerosis and survival, production and secretion of growth factors, and activity [67]. ECM constituents [75]. Tis research demonstrated for the frst time that the maggot was capable of producing a human 14. Discussion growth factor from the transgene expression systems. It sheds a light on the combination of MDT and genetic engineering Many people may have their doubts about using maggots as to a new clinical application. Predictably, genetically modifed medicine. However, they should keep in mind that a large maggot will be engineered to express and secrete diverse number of drugs come from nature’s pharmacy. For example, heterologous proteins which are more benefcial to wound hirudin, a bivalent direct thrombin inhibitor extracted from healing in the future. leeches, is approved for heparin-induced thrombocytope- nia and acute coronary syndromes [70]. In addition, anti- 15. Conclusion infammatory protein-2, a protein secreted by hookworms, was found to serve as a novel curative therapeutic for allergic Te “medicinal maggot,” which is the core of MDT, demon- asthma and potentially other infammatory diseases [71]. strates possessing diverse properties. We summarize the Similarly, maggot is also a real treasure-trove of raw mate- current scientifc laboratory evidences of the antimicrobial, rials for clinical application. MDT was used extensively in antibioflm, anti-infammatory, and wound healing activities hospitals during the 1930s and 1940s. Te application of MDT for MDT, particularly active ingredients (e.g., lucifensin, nearly disappeared from the early 1940s to 1980s because of Seraticin, Chymotrypsin, DNAse, and unsaturated fatty acid) the invention of penicillin and better surgical techniques. within maggots involved in these benefcial efects. Te devel- However, with the dilemma of antibiotic resistance and a opments of recombinant and transgenic bioactive molecules diminishing arsenal of efective antibiotics, MDT revived as from the maggot are both challenges and opportunities a potent, universal therapy for chronic, intractable wounds for increased treatment acceptance and improved outcome. infected with MRSA and has been proven efective in cleaning Perhaps one day in the future, these molecules may surpass andhealingwoundsthatrespondpoorlytootherformsof the use of the maggot itself in the treatment of chronic conventional treatments. Even more surprising is that the and refractory wounds. Sincerely, we hope to form a more combinations of MDT and antibiotics enhance antibacterial detailed theory foundation from which great eforts towards activity. In addition, maggot produces a variety of active therapeutic agents of maggots for medical purposes can be ingredients, such as lucifensin with antibacterial activity, made. lucimycin with antifungal bioactivity, and chymotrypsin with proteolyticactivity.Forconvenience,relevantactivitiesand Conflicts of Interest responsible components/mechanisms within maggots are summarized in Table 1. Nonetheless, many other molecules Te authors declare no conficts of interest. with benefcial activities still need to be further identifed and characterized. For this purpose, next generation sequencing Authors’ Contributions could be available for researchers to focus their attention on genomic information. For example, Franta et al. applied Mingzhi Song and Shouyu Wang contributed equally to this next generation sequencing to analyze the transcriptome manuscript. of maggots and identifed 577 clusters representing 185 peptidase species [72]. Although detailed function of these putative peptidases was not provided, it still produced enough Acknowledgments data for the further identifcation of bioactive proteins within Tis study was supported by National Natural Science maggots. Foundation of China (no. 81573734) and Natural Science All these molecules could be potential candidates for Foundation of Liaoning Province of China (no. 2015020310). therapy, and hence high purifcation and mass production are top priority for wide use. As is known to all, it is almost impossible to obtain a large number of highly purifed References proteins from original organic sources. Recombinant protein [1] R. A. Sherman, “Mechanisms of maggot-induced wound heal- technology has quite acceptably solved this problem in ing: what do we know, and where do we go from here?” several researches. 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