CN111375053B

CN111375053B - Preparation method and application of oral recombinant human lactoferrin sericin nanoparticle

Info

Publication number: CN111375053B
Application number: CN202010185707.XA
Authority: CN
Inventors: 王峰; 夏庆友; 许胜�; 赵萍
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2020-03-17
Filing date: 2020-03-17
Publication date: 2023-09-05
Anticipated expiration: 2040-03-17
Also published as: CN111375053A

Abstract

The invention provides an effective and safe preparation method and application of oral recombinant human lactoferrin sericin nano-particles, which can improve the intake efficiency of rhLF cells, inhibit the inflammatory generation of macrophages induced by LPS and treat DSS-induced acute colitis. The rhLF transgenic silkworm cocoon is taken as a raw material, and the rhLF-containing sericin nanoparticle is prepared through sericin extraction, dialysis and ethanol induction, is directly used for oral treatment of colonitis, is suitable for large-scale production, and has the advantages of simple operation and high application value.

Description

Preparation method and application of oral recombinant human lactoferrin sericin nanoparticle

Technical Field

The invention is applied to the technical field of biological medicine, and in particular relates to a preparation method and application of oral recombinant human lactoferrin sericin nano-particles.

Background

Lactoferrin is a multifunctional, naturally occurring immune glycoprotein that has been found to have biological activity in re-immunization, antibacterial, antitumor, antioxidant, stimulating cell growth, and the like. Lactoferrin is widely present in exosomes such as milk, tears, pancreatic juice, bile, and semen, and is mainly stored by neutrophil secondary particles. Lactoferrin is present in very low levels in normal physiological conditions and increases significantly with infection. The detection of abundant lactoferrin receptors by researchers in activated B and T lymphocytes, monocytes/macrophages, testicular brush border cells, platelets, novacells, and the like means that lactoferrin has a variety of biological functions. The activity of participating in iron ion transport, regulating immunity, resisting bacteria, resisting tumor, resisting oxidation, stimulating cell growth and the like has been verified. Furthermore, lactoferrin may affect the release of inflammatory factors such as IL1 beta, IL12, IL6, TNF-alpha, etc. by inhibiting the activation of MAPK/NF- κβ signaling pathways. Research has shown that a large number of inflammatory factors secreted by macrophages, including TNF- α, IL6 and IL-12, play a critical role in the development of UC, and thus lactoferrin is a potential drug for the treatment of colitis.

Sericin is one of main components of silkworm silk, has good hydrophilicity, biocompatibility and degradability, is a very useful biological material, is used for intestinal repair by taking sericin reinforced bacterial cellulose as a tissue engineering bracket, can effectively inhibit the growth of bacteria and fungi by taking hydrogel loaded with nano silver consisting of sericin and polyvinyl alcohol, can promote the healing of chronic wounds by taking soft agar sericin gel as a biological material for in vivo ischemia myocardial repair, can obviously inhibit the growth of mouse melanoma by taking injectable hydrogel prepared from sericin/levorotatory anhydride, can obviously promote the proliferation of cells by taking hydrogel loaded with FGF1 outside the hydrogel body by taking 3D printing technology to prepare sericin film for promoting the healing of wounds, and can reduce the toxicity of the doxorubicin by taking sericin nanoparticles.

Inflammatory bowel diseases, including crohn's disease and ulcerative enteritis, are the most common inflammatory bowel diseases in developed countries, with the major conditions being abdominal pain, diarrhea, rectal bleeding and weight loss. In recent years, the incidence of ulcerative enteritis has been on the rise worldwide. However, in existing therapeutic drugs such as lactose sterols, aminosalicylate, infliximab, etc., are used only for the alleviation of symptoms of clinical ulcerative enteritis, and these drugs have low solubility, poor stability and side effects, the quality of life of patients is still adversely affected.

In the treatment of UC, rectal administration is a common treatment modality, which, although it allows the drug to reach the lesion rapidly, is complex to handle for the patient himself, and the administration conditions are severe, especially outside the door, thus reducing patient compliance. Among the many modes of administration, oral administration is more acceptable to patients. Oral protein drugs are often susceptible to degradation by gastric acid or digestive enzymes, and drug delivery systems can assist proteins in traversing complex digestive environments, with drug delivery systems being an effective way to increase protein utilization. Current drug delivery systems for UC treatment include liposomes, micelles, mesoporous silica nanoparticles, hydrogels, etc., each of which has characteristics such as enhanced drug solubility by enhanced epithelial cell penetration and retention, enhanced bioavailability by targeting effects, etc., and although these drug systems are effective, complex manufacturing processes, low drug loading clearly limits their development to clinical applications. Therefore, it is particularly important to study an effective and safe sericin oral drug.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides an effective and safe preparation method and application of oral recombinant human lactoferrin sericin nano-particles, which can improve the intake efficiency of rhLF cells, inhibit the inflammatory generation of macrophages induced by LPS and treat DSS-induced acute colitis.

The invention solves the technical problems by adopting the following technical scheme:

the preparation method of the oral recombinant human lactoferrin sericin nanoparticle comprises the following steps: the recombinant human lactoferrin transgenic silkworm is taken as a raw material, urea water bath extraction and dialysis are adopted to obtain sericin solution, absolute ethyl alcohol is used for induction, air drying and resuspension are carried out to obtain the oral recombinant human lactoferrin sericin nano-particles.

Further, the conditions for extracting urea in a water bath are as follows: the crushed silkworm cocoon powder and urea solution are extracted for 1h in a water bath kettle with the temperature of 80 ℃ by using urea according to the bath ratio of 10 mg/mL.

Further, the induction process is as follows: the silk gum solution obtained by dialysis is added dropwise into absolute ethyl alcohol at the flow rate of 100 mu l/min for induction.

Further, the ratio of the sericin solution to the absolute ethyl alcohol is 1:4.

Further, the air drying and resuspension processes are as follows: and (3) placing absolute ethyl alcohol containing the sericin nano particles on an ultra-clean workbench for air drying after the induction is finished, and then, re-suspending and collecting the sericin nano particles by using normal saline.

Further, the diameter of the sericin nanoparticle is less than 200nm.

An application of the oral recombinant human lactoferrin sericin nanoparticle in the preparation of medicines for preventing or/and treating colonitis.

Furthermore, the oral recombinant human lactoferrin sericin nanoparticle can inhibit inflammatory generation of macrophages induced by LPS.

Furthermore, the oral recombinant human lactoferrin sericin nanoparticle can treat DSS-induced acute colitis.

Further, the oral recombinant human lactoferrin sericin nanoparticle reduces secretion of TNF- α, IL2, IL6 and IL 12.

The medicine for preventing or/and treating colonitis comprises recombinant human lactoferrin sericin nano-particles and pharmaceutically acceptable auxiliary materials.

The oral recombinant human lactoferrin sericin nanoparticle can be singly used or can be matched with other medicines for simultaneous use or can be prepared into a compound preparation with other medicines for use when preventing or/and treating colonitis, and the purpose of preventing or/and treating colonitis can be achieved.

The pharmaceutically acceptable auxiliary materials refer to various conventional auxiliary materials such as diluents, adhesives, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorbing materials and the like which are required when different dosage forms are prepared, and any common oral preparation such as granules, powder, tablets, capsules, pills, oral liquids, decoctions, dripping pills and the like can be prepared by a conventional preparation method.

Compared with the prior art, the invention has the beneficial effects that:

1) The invention establishes an effective and safe recombinant human lactoferrin sericin nanoparticle, inhibits the generation of inflammatory by macrophages induced by LPS and treats DSS-induced acute colitis, is directly used for oral treatment of colitis, is suitable for large-scale production, and has simple operation and high application value.

2) The recombinant human lactoferrin sericin nanoparticle provided by the invention has negative charges, the negative charges of sericin can be combined with the positive charges of inflammatory parts, and the carried medicine can be released to play a role, so that the intake efficiency of rhLF cells can be improved, the inflammatory generation of macrophages induced by LPS can be inhibited, and the DSS induced acute colitis can be treated.

Drawings

Fig. 1 is a graph showing the preparation and characteristic analysis of sericin nanoparticles in the preparation method and application of the oral recombinant human lactoferrin sericin nanoparticles according to the present invention.

Wherein A is a sericin nanoparticle aperture detection graph, B is a rhLF cumulative release characteristic detection graph in PBS with different pH values, C is a potential detection graph of WT-nano and rhLF-nano zeta, D is a SEM graph of WT-nano morphology, E is a TEM graph of WT-nano morphology, F is a secondary structure analysis graph of WT-nano and rhLF-nano morphology, G is a SEM graph of rhLF-nano morphology, and H is a TEM graph of rhLF-nano morphology.

Fig. 2 is a schematic diagram of rhLF cell uptake detection in rhLF-nano in the preparation method and application of an oral recombinant human lactoferrin sericin nanoparticle according to the present invention.

FIG. 3 is a diagram showing the analysis of inhibiting LPS induced raw264.7 cells from producing inflammatory in the preparation method and application of the oral recombinant human lactoferrin sericin nanoparticle.

Fig. 4 is a schematic diagram of a preparation method of an oral recombinant human lactoferrin sericin nanoparticle and detection of colon structure by mouse hematochezia and HE staining in application of the oral recombinant human lactoferrin sericin nanoparticle.

Fig. 5 is a diagram of the detection and analysis of the preparation method and application of the oral recombinant human lactoferrin sericin nanoparticle for treating acute colitis in mice.

Wherein A is a comparative graph of colon length of each group, B is a comparative graph of colon length of each group, C is a comparative graph of colon weight, D is a graph of colon tissue score, E is a comparative graph of spleen weight of a mouse, F is a graph of IL2 content of colon tissue, G is a graph of IL6 content of colon tissue, H is a graph of IL10 content of colon tissue, I is a graph of MPO content of colon tissue, and J is a graph of Disease Activity Index (DAI) of a mouse.

FIG. 6 is a schematic diagram showing the immunofluorescence detection of rhLF-nano inhibition of p65 protein entering the nucleus in the preparation method and application of the oral recombinant human lactoferrin sericin nanoparticle.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to the accompanying drawings and specific examples, wherein the experimental methods without specific conditions are generally according to conventional conditions. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.

Experimental materials used in the examples of the present invention: recombinant human lactoferrin cocoons (rhLF) and wild cocoons (WT) were obtained from the laboratory rearing recombinant human lactoferrin transgenic silkworm strain and the D9L strain. The silkworms are fed with artificial feed in an artificial climate box at 25 ℃. Mouse macrophage Raw264.7 was maintained in laboratory and cultured in DMEM medium containing 10% (v/v) fetal bovine serum (FBS, gibco) at 37℃under 5% CO ₂ . The experimental mice were Balb/C, females, weighing about 22g, and were housed in a barrier environment IVC feeding system.

The conventional culture medium, reagent buffer and the like used in the molecular cloning process of the embodiment of the invention are configured by referring to the section of the laboratory conventional reagent preparation method in TaKaRa commodity catalog in the third edition of molecular cloning experiment guide.

Example 1: preparation of sericin nanoparticles

rhLF cocoons and WT cocoons were obtained from our laboratory by raising rhLF silkworm strain and D9L silkworm strain, and cocoons were kept at room temperature before use. WT and rhLF silkworm strains are raised by fresh mulberry leaves to obtain WT and rhLF cocoons, and the cocoons are stored at room temperature before being used. The silkworm cocoons are frozen by liquid nitrogen and then crushed into silkworm cocoon powder by a high-speed crusher. Weighing a certain amount of silkworm cocoon powder, extracting with 50mM Tris-HCl containing urea with pH of 8.0M at a bath ratio of 10mg/mL in a water bath at 80 ℃ for 1h, filtering with gauze, removing undissolved silk fibroin to obtain a silk gum solution, centrifuging the silk gum solution at 10000rpm at 25 ℃, and collecting the supernatant for later use. The WT sericin solution and the rhLF sericin solution are dialyzed with deionized water with the molecular weight of 10kDa, the deionized water is changed every 12 hours, the total dialysis is carried out for 4 days, and the sericin solution is collected for standby after the dialysis is completed. The sericin solution was induced by dropwise addition to a beaker containing absolute ethanol at a flow rate of 100 μl/min by a peristaltic pump until the sericin solution and absolute ethanol ratio was =1: and 4, magnetically stirring to promote generation of nanoparticles, placing absolute ethyl alcohol containing the sericin nanoparticles on an ultra-clean workbench for air drying after induction, and finally, resuspending and collecting the sericin nanoparticles with normal saline, wherein the sericin nanoparticles are placed in a refrigerator at 4 ℃ for later use. Wherein the sericin nano-particle prepared by the WT silkworm cocoon is named as WT-nano, and the nano-particle prepared by the rhLF silkworm cocoon is named as rhLF-nano.

Example 2: characterization of sericin nanoparticles

Referring to fig. 1, pore diameter, secondary structure, release characteristics and the like of recombinant human lactoferrin sericin nanoparticles are detected, and the specific detection method and the specific detection result are as follows:

1. fourier transform infrared absorption spectrometer (FTIR) analysis

The secondary structure of the sericin nanoparticle was analyzed by FTIR (Nicolet iN10 instrument, siemens, USA) with a spectral acquisition range of 800-4000cm-1, followed by secondary structure analysis by the amide I peak-splitting method of 1640-1610cm ^-1 ，1650-1640cm ^-1 ，1660-1650cm ^-1 ，1700-1660cm ^-1 Representing β -sheet, random coil, α -helix, and β -turn, respectively, more than 30 independent samples were used for FTIR analysis.

Referring to fig. 1A and 1F, the average particle diameters of the prepared sericin nano particles are measured to be within 200nm, the average particle diameter of the rhLF-nano particles is 123.30 +/-10.42 nm, the PDI index is 0.168, the average particle diameter of the WT-nano particles is slightly larger than the average particle diameter of the rhLF-nano particles, but the difference is not obvious, and the average particle diameter of the WT-nano particles is 147.25 +/-9.85 nm and the PDI is 0.144. The secondary structure results show that the beta-sheet content in the WT-nano and rhLF-nano is relatively high, namely 42.58% and 46.63% respectively.

2. Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) analysis

After nanoparticle preparation, freeze-dried, lyophilized recombinant human lactoferrin sericin nanoparticles were placed on a dedicated stage of a scanning electron microscope with the observation face facing up, and lyophilized hydrogels were plated with a layer of platinum under vacuum, followed by observing the structure of each sample at room temperature under an accelerated operating voltage of 10kV and photographing. The nanoparticles were dissolved in an aqueous solution, followed by ultrasonic dispersion and observation on an engine.

Referring to fig. 1D, 1E, 1G and 1H, SEM and TEM morphology detection is carried out on the sericin nano particles, so that WT-nano and rhLF-nano are similar to spheres, and finer particles are adhered on the surfaces of the particles.

3. Analysis of rhLF Release Properties in rhLF-nano

The pH of the 0.1mM PBS buffer was adjusted to 5.5, 6.8, 7.4 with 0.1M hydrochloric acid or 0.1M sodium hydroxide, respectively; 100 μl rhLF-nano was placed in 900 μl of PBS with different pH, each sample was placed on a shaking table, the supernatant was centrifuged at 37deg.C and 100rpm and at certain time points (1, 2, 4, 6, 12, 18 and 24 h), the supernatant was transferred to a new centrifuge tube, stored at-40deg.C before detection, then 1ml of fresh PBS with different pH was added to the tube and the nanoparticle was continuously processed under the same conditions, and eliza was performed to detect rhLF content after all samples were taken out. Each group was replicated three times.

Referring to FIGS. 1B and 1C, zeta potential measurements of sericin nanoparticles showed that WT-nano and rhLF-nano were negatively charged, and at low pH (pH 1.25, pH 2.5), WT-nano and rhLF-nano nanoparticles showed weak charge, pH was increased to 5.5, the charge of the nanoparticles was suddenly increased, and then pH was increased, and the charge of the nanoparticles was not greatly changed. Good drug release performance is one of important indexes for evaluating the superiority of a drug delivery system, and the results of drug release behavior detection of rhLF-nano in buffer systems with different pH values (5.5, 6.8 and 7.4) show that the rhLF-nano has good release performance under the conditions of pH5.5, 6.8 and 7.4, the release rates of the rhLF-nano are basically consistent, more than 60% of the rhLF-nano is released in the first 6 hours, and the release is basically completed after 24 hours, so that the rhLF-nano has important significance for the dissolution and volatilization functions of the rhLF-nano.

Example 3: detection of rhLF cell uptake in rhLF-nano

RAW264.7 macrophages are added into 96-well plates at a density of 5X 104 cells/well, cultured for 12 hours in DMEM medium containing 10% FBS, 90. Mu.l/well of the medium is replaced after the cells are attached, then 10. Mu.l of WT-nano, rhLF-nano and rhLF-STD solutions are added respectively, the medium is removed at a certain time point (1, 2 and 4 hours), the medium is washed three times with cold PBS, then 4% paraformaldehyde is added to fix 10min,Triton X100 for cell perforation, anti-rhLF antibodies and immunofluorescence secondary antibodies are used for cell rhLF labeling, and after the labeling is completed, the cells are observed and photographed under a fluorescence microscope. Each group was replicated three times.

To investigate whether rhLF was active by macrophage endocytosis, cells were co-cultured with WT-nano, rhLF-nano and rhLF-STD, respectively, and the effect of rhLF endocytosis to cells was examined with anti-rhLF specific antibodies. Referring to FIG. 2, no red fluorescence was observed in the negative control WT-nano co-culture with cells, and a significant red fluorescence signal was detected around the nuclei after 1h of co-incubation of rhLF-nano with cells, indicating that a small amount of rhLF was taken up by macrophages. In addition, co-culture with macrophages for 2h and 4h, the red fluorescent signal enhancement indicates that the absorption of rhLF by macrophages has a time effect. The rhLF-STD solution treated cells were also clearly detectable for rhLF-specific red fluorescence, but the red fluorescent signal points were significantly less compared to the rhLF-nano treated group, especially at the 1h and 2h treatment time points. The results show that the rhLF can be effectively absorbed by macrophages, the absorption efficiency has time dependence, and the absorption efficiency of rhLF-nano is stronger than that of rhLF-STD, and the results provide a material basis for enhancing the anti-inflammatory activity of macrophages.

Example 4: in vitro inhibition of LPS-induced macrophage production is an inflammatory.

RAW264.7 macrophages were added to 96-well plates at a density of 5X 104 cells/well, cultured in DMEM medium containing 10% FBS for 12 hours, after cell attachment, the complete medium was replaced with medium containing WT-nano, rhLF-nano and rhLF-STD solutions, respectively, and the remaining cells were added with LPS (200 ng/ml) except for the negative control group and incubated for 12 hours. Cell culture media were collected after incubation was completed and assayed for the content of proinflammatory factors using TNF- α, IL2, IL6 and IL12 Eliza kit. LPS stimulated macrophages were used as positive controls and cells not treated with LPS were used as negative controls, 3 replicates per group.

As shown in FIG. 3, the levels of pro-inflammatory factors in the macrophage supernatant medium treated with LPS were much higher than in the negative control group (CK). In sharp contrast to LPS and WT-nano treated groups, rhLF-nano treated cells effectively reduced secretion of TNF- α, IL2, IL6 and IL12, with similar effects as the positive control group (rhLF-STD). These results indicate that WT-nano has no anti-inflammatory activity in vitro, whereas rhLF-nano has a significant inhibitory effect on LPS-induced RAW264.7 macrophage inflammatory response.

Example 5: in vivo treatment of DSS-induced colitis

6-8w female Balb/C mice are taken for the experiment, and 3.5% Dextran Sodium Sulfate (DSS) with the molecular mass of 36-50kDa is added into daily drinking water of the rest mice except the CK group for continuous treatment for 8 days, and physiological saline (S), WT-nano, rhLF-nano and rhLF-STD are respectively given daily while DSS treatment is carried out, and are respectively defined as an S group, a WT-nano group, an rhLF-nano group and an rhLF-STD group. The mice were photographed after the last dosing treatment to record the defecation, and the mice were sacrificed and weighed for subsequent experiments with spleen and colon.

1. Histological assessment of H & E staining and colitis

The colon tissues of each group of mice were fixed in 4% paraformaldehyde, embedded in paraffin after ethanol gradient dehydration, sectioned in paraffin, stained with H & E, followed by observation of the colon structure under a microscope and recording by photographing. Histological scores will be made from 3 parameters: severity of inflammation, crypt injury and ulcers, each index was scored as follows: inflammation: the lamina propria is free of inflammatory cells (0 min), the number of lamina propria granulocytes is increased (1 min), inflammatory cells are infused into submucosa (2 min), and inflammatory infiltrates are prolonged across the wall (3 min); crypt damage: complete crypt (0 min), base one third (1 min), base two thirds (2 min), whole crypt (3 min), epithelial surface changes with erosion (4 min), confluent erosion (5 min); ulcers: no ulcers (0 points), 1 or 2 ulcers (1 point), 3 or 4 ulcers lesions (2 points), combined or extensive ulcers (3 points). The sum of these values gives a score in the range of 0 to 11 points. Three sections of colon were scored for each animal.

Referring to fig. 4, by observing that the CK group mice have no hematochezia phenomenon, the mice with gastric lavage physiological saline (S) and WT-nano present different degrees of hematochezia, the blood attached to the fur near the anus is clearly visible, the anus cleanliness of the mice with gastric lavage rnLF-nano and rhLF-STD is similar to that of the CK group mice, although the two groups mice also present hematochezia phenomenon, the symptoms are lighter than those of the S group, and the rhLF-nano has better treatment effect from the initial observation result of the appearance form.

Histological observation by HE staining showed that DSS severely destroyed the crypt and goblet structures of the colon of mice (group S) and that immune cells were abnormally aggregated, and that small portions of local immune cell proliferation were observed in the tissue of mice in the rhLF-nano and rhLF-STD treated groups, but that goblet and crypt structures were clearly discernable and exhibited similar histological structures as those of group CK. By carrying out histological scoring on the colon injury scoring system, the score of the CK group is 0, the average score of the colon injury of the WT-nano group is 2.4, the average score of the colon injury of the rhLF-nano group is only 0.8, and the score of the rhLF-nano group is found to be closer to the score of the CK group, so that the method has a better immune effect.

2. Mouse Disease Activity Index (DAI) score

The DAI score ranges from 0 to 4 points with the following criteria:

the DAI score integrates the parameters of mouse body weight, blood and stool concentration, etc. Daily observations recorded the health of the mice, and the recorded scores indicated that the DAI score of the mice in the S-treated group increased progressively with increasing DSS treatment time, see FIG. 5I, and that the DAI was significantly lower in the rhLF-nano and rhLF-STD treated groups at the end of the experiment than in the S group.

3. Determination of MPO, IL2, IL6 and IL10 content in colon tissue

A colon tissue homogenate was prepared from the distal colon tissue of each group of mice using a tissue homogenate kit, and then MPO content determination was performed on each group of colon homogenate tissue using an MPO Eliza kit, and one unit of MPO activity was defined as the amount of degrading 1. Mu. Mol peroxidase per minute, and the result was expressed as absorbance per g protein. IL2, IL6 and IL10 were each assayed using the corresponding Eliza kit. Each group of

Referring to fig. 5A, 5B, 5C, 5E, dissected mice removed the colon, and DSS was found to significantly shorten the length of the mouse colon (S group), which was only 75% of healthy mice (CK group), with rhLF-nano treated mice significantly longer than S group. Clearly, the colon length of rhLF-nano and rhLF-STD treated mice were not statistically different from that of the CK group mice. Colon weighing statistics of each group of mice show that the colon weight of the S group of mice is the lightest and is 600.6+/-45.75 mg, the rhLF-nano and rhLF-STD treatment groups are 32.35 percent and 30.42 percent heavier than the S group respectively, and the colon weight of the S group are not significantly different from the colon weight of the CK group. The spleen weights of the mice in each group are contrary to the trend of the colon length and weight of the mice, which means that the intragastric rhLF-nano does not cause the spleen of the mice to generate swelling overstimulation reaction.

Referring to FIGS. 5F-5I, the colon of the mice was homogenized and tested for inflammatory factor expression, and the colon IL6, IL12 expression was significantly higher in group S mice than in group CK. However, rhLF-nano and rhLF-STD treatment effectively reduced the expression of inflammatory factors IL6 and IL12 in colon tissue. Since IL10 plays an important role in inhibiting inflammatory responses, we examined IL10 expression in colon tissue of each group of mice simultaneously. We detected that rhLF-nano treated mice had up-regulated IL10 expression from group S, although there was no statistical difference between them. MPO is generally one of the indicators for evaluating the degree of inflammation, and is secreted by activated neutrophils, as shown in fig. 5J, the colon activity of mice in group S is significantly increased compared to that in group CK, and the MPO activity in the remaining treated group is significantly lower than that in group S, notably, the rhLF-nano group exhibits lower MPO activity than WT-nano, indicating that rhLF is more efficient for decreasing MPO activity.

4. Immunofluorescence detection of colon p65 protein entry into cell-check assay

In order to detect the potential action path of rhLF-nano, an immunofluorescence method is used for detecting the nuclear entering condition of the colon p65 protein of a mouse, paraffin sections are dewaxed conventionally, anti-p65 fluorescent antibodies are used for marking the p65 protein and DAPI is used for marking cell nuclei, finally immunofluorescence observation and photographing are carried out under a fluorescence confocal microscope, and finally software ZEN 3.0 is used for analysis.

Typically the onset of inflammation can activate the passage of cell p65 from the cytoplasm into the nucleus. The expression of p65 in colon tissue nuclei was observed by laser confocal microscopy, and as shown in fig. 6, each group of mouse colon cells showed specific red fluorescence, however, their expression on nuclei was greatly different. The red fluorescence of the P65 of the CK group mice is not overlapped with the blue fluorescence basically, which shows that the P65 does not enter the cell nucleus, and the blue fluorescence of the S group nuclear WT-nano treatment group mice is overlapped with more red fluorescence nuclei, and obviously, the overlapping condition of the two fluorescence signals of the rhLF-nano treatment group and the rhLF-STD treatment group is obviously less. Taken together, rhLF-nano is effective in treating DSS-induced acute colitis, which potentially acts by inhibiting pathway NF- κb.

The positive control rhLF-STD solution used in the present invention was a commercially available standard solution.

The above description is merely a preferred embodiment of the present invention, and the scope of the present invention is not limited to the above examples. Any modification, equivalent replacement, improvement, etc. made by those skilled in the art without departing from the technical idea of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of oral recombinant human lactoferrin sericin nano-particles in preparing a medicament for preventing or/and treating colonitis, wherein the pH value of the recombinant human lactoferrin sericin nano-particles is 5.5-7.4;

the preparation method of the recombinant human lactoferrin sericin nanoparticle comprises the following steps: taking recombinant human lactoferrin transgenic silkworms as raw materials, extracting crushed silkworm cocoon powder and urea solution in a water bath kettle of 80 ℃ with urea for 1h according to a bath ratio of 10mg/mL, obtaining a sericin solution after dialysis, dropwise adding the sericin solution obtained by dialysis into absolute ethyl alcohol at a flow rate of 100 mu l/min for induction, wherein the volume ratio of the sericin solution to the absolute ethyl alcohol is 1:4; placing absolute ethyl alcohol containing sericin nano particles after induction is finished in an ultra-clean workbench for air drying, and then re-suspending and collecting the sericin nano particles by using normal saline; the diameter of the sericin nano-particles is smaller than 200nm.

2. The use according to claim 1, wherein: the medicine prepared from the oral recombinant human lactoferrin sericin nanoparticle can inhibit inflammatory of macrophages induced by LPS.

3. The use according to claim 1, wherein: the medicine prepared from the oral recombinant human lactoferrin sericin nanoparticle can treat DSS-induced acute colitis.

4. The use according to claim 1, wherein: the oral recombinant human lactoferrin sericin nanoparticle reduces secretion of TNF-alpha, IL2, IL6 and IL 12.