The Expression Of Recombinant Human Lactoferrin In Silkw Orm And Its Treatment On Ulcerative Colitis By Oral Administration

Lactoferrin (LF), a kind of iron-binding glycoprotein with a variety of biologicalactivities and functions, mainly exists in the mammalian milk and has become ahigh-profile drug development target in the areas of antiviral, antitumor,anti-inflammatory and so on. At present, LF for drug research mainly comes from thenatural LF extracted from mammalian milk and recombinant LF obtained by generecombination technique, both need to be purified and concentrated. The lowproduction, complex process and high cost limit the drug development and massproduction.Based on the advantages of silkworm with abundant resources in our country,easy breeding, modification of expression product after translation, and the safe andedible characteristics of the pupa, combining with the anti-inflammatory andimmunoregulation functions of LF, in this study, the recombinant human lactoferrin(rhLF) was expressed in silkworm pupa infected with recombinant baculoviruscontaining hLF gene, and the oral treatment on ulcerative colitis (UC) in mice wasconducted directly by freeze-dried pupa powder without purification, aiming toexplore a new and economic method for the treatment on UC. First of all, the pupainfected with recombinant baculovirus vBm-hLF successfully expressed the rhLF,with a molecular weight of about80kDa and a high-level expression up to0.38mg/mL supernatant of pupa homogenate and1.2mg/g pupa powder afterlyophilization. Antibacterial activity in vitro against E.coli TG1showed that the pupapowder containing rhLF inhibited the growth or even killed E.coli TG1, suggesting itsantibacterial activity. For safety evaluation, acute toxicity test was conducted in micewith a maximum dosage (10g/kg) of rhLF pupa powder. No obvious toxic effectswere found during the observation period and the major organs of mice were notobviously impaired, indicating that rhLF pupa powder was low-toxic to mice by oraladministration.After determining the biological activity and safety of rhLF pupa powder, weexplored the therapeutic effect on mice with acute UC. Mice with acute UC induced by5%DSS was gavaged with high, medium and low (5,2.5,1.25g/kg) doses of rhLFpupa powder respectively for treatment, and salazosulfapyridine (SASP) and normalpupa powder was respectively used as the positive and negative controls. The resultshowed that rhLF pupa powder obviously alleviated the inflammatory response andcolon injury, lowered the DAI score and the levels of MPO and OPN, indicating thatrhLF pupa powder performed a certain therapeutic effect on mice with acute UC. Inorder to further explore the therapeutic mechanism of rhLF pupa powder, the serumlevels of IL-1α, IL-4, IL-6, IL-10in mice was tested by ELISA, the results of whichshowed a significant decrease in the proinflammatory cytokines IL-1α, IL-6and adistinct increase in the anti-inflammatory cytokines IL-4, IL-10after treated by rhLFpupa powder. Thus infer that the promotion for the recovery of colon tissueinflammation and the effectiveness on UC treatment of rhLF pupa powder is mainlyachieved by adjusting the balance of cytokines network.In this study, rhLF with biological activities was successfully expressed in thesilkworm pupa, and the therapeutic effect and mechanism of rhLF pupa powder onacute UC in mice were preliminarily explored, which will lay a solid foundation forthe future clinical research and drug development.