CN114177294A

CN114177294A - Application of chlamydia protein Pgp3 in preparation of medicament for inhibiting salpingitis

Info

Publication number: CN114177294A
Application number: CN202111528170.3A
Authority: CN
Inventors: 侯淑萍
Original assignee: Tianjin Medical University General Hospital
Current assignee: Tianjin Medical University General Hospital
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-03-15

Abstract

The invention provides application of chlamydia protein pgp3 in preparation of a medicament for inhibiting salpingitis. Experimental research proves that pgp3 can be specifically combined with TNF-alpha, and can effectively inhibit TNF-alpha mediated host cell apoptosis after combination, so that the growth cycle of chlamydia in the host cell is ensured; pgp3 induces an inflammatory response in fallopian tube epithelial cells, which is synergistically enhanced upon binding of TNF- α.

Description

Application of chlamydia protein Pgp3 in preparation of medicament for inhibiting salpingitis

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of chlamydia protein Pgp3 in preparation of a medicament for inhibiting salpingitis.

Background

Chlamydia trachomatis is a gram-negative obligate intracellular parasitic prokaryote that needs to be parasitic in host cells for growth and reproduction. Vaccination may be more effective than other biomedical interventions in controlling chlamydial infection, but vaccines that are effective in inducing mucosal and systemic protective responses have not been well developed due to the immunological properties of the reproductive tract and the infectious properties of chlamydia against mucosal epidermal cells.

The inflammatory reaction is a common reaction after chlamydia trachomatis infects genital tracts, which is usually manifested as dilation and hydrops of the fallopian tubes and infiltration of neutrophils, and the long-term inflammatory reaction can cause fibrosis and blockage of the fallopian tubes and further can cause tubal infertility. The pathogenicity of the oviduct of chlamydia depends on the one hand on the ability of the chlamydia to ascend infections and on the other hand on the ability of the chlamydia to induce inflammatory responses. Although it has been confirmed in vitro and in vivo experiments: chlamydia trachomatis infection can cause tubal lesions, but the pathogenic mechanism thereof has not been clarified at present. To survive and cause disease, many bacteria have their own strategies to combat the attack of antimicrobial peptides, such as secretion of agents that bind to or degrade antimicrobial peptides. In the case of chlamydia, the plasmid and the protein encoded by it are a well-defined factor that can protect against antimicrobial peptides. Although chlamydia has different serotypes, its plasmid structure is highly conserved, with pgp3 being the most important of all proteins encoded by the plasmid, and the only protein secreted into the cytoplasm. Current studies indicate that pgp3 is an important virulence factor for chlamydia.

pgp3 is encoded by a chlamydia plasmid and is the only protein efficiently secreted into the cytosol of the cell, accumulates as a trimer into the cytosol of the host cell at the late stages of infection, and upon rupture of the host cell, pgp3 is released extracellularly prior to EB, directly contacting the host's internal environment.

However, the current state of research is still unclear as to how pgp3 causes salpingitis.

Disclosure of Invention

In view of the above, the invention aims to overcome the defects in the prior art and provides an application of chlamydia protein Pgp3 in preparing a medicament for inhibiting salpingitis.

In order to achieve the purpose, the technical scheme of the invention is realized as follows:

in a first aspect, the invention provides the use of chlamydia protein pgp3 in the manufacture of a medicament for the treatment of infertility in the fallopian tube.

Preferably, the infertility of fallopian tube is infertility caused by salpingitis.

Preferably, the salpingitis is a tubal lesion caused by chlamydia protein pgp 3.

Preferably, the medicament inhibits the development of salpingitis by inhibiting the binding of the chlamydia protein Pgp3 to the tumor necrosis factor TNF- α.

In a second aspect, the present invention provides an inflammation inducing agent comprising chlamydia protein Pgp3, which induces inflammation by inducing the binding of chlamydia protein Pgp3 to tumor necrosis factor TNF- α.

Preferably, the inflammation is inflammatory lesions of the oviduct induced by chlamydia protein pgp3, stimulating secretion of IL-6 and IL-8 by oviduct epithelial cells.

Compared with the prior art, the invention has the following advantages:

based on the research basis of the prior art, the chlamydia plasmid coding protein pgp3 is selected as a research object, a 6-week-old magnetic mouse model is used as a tool, and the protein is proved to cause the salpingitis reaction in a mode of injecting His-pgp3 into an ovarian cyst, and the inflammatory reaction degree is in direct proportion to the protein concentration. Binding of pgp3 to TNF-. alpha.was confirmed by amino acid conjugation. In vitro cell culture demonstrated that pgp3 was effective in inhibiting TNF- α mediated apoptosis of host cells and synergistically stimulating secretion of IL-6 and IL-8 by fallopian tube epithelial cells.

The invention clarifies the specific mechanism of oviduct lesion caused by chlamydia trachomatis infection. Experimental research proves that pgp3 can be specifically combined with TNF-alpha, and can effectively inhibit TNF-alpha mediated apoptosis of host cells after combination, so that the chlamydia can complete a growth cycle in the host cells; pgp3 induces an inflammatory response in fallopian tube epithelial cells, which is synergistically enhanced upon binding of TNF- α.

Drawings

FIG. 1 is a mouse oviduct specimen (a is blank group, b is negative control group, c is low concentration group, d is high concentration group);

FIG. 2 is a HE stained section 40X of the oviduct or ovary of each group of mice (a is a high concentration group, b is a blank group, c is a low concentration group, d is a negative control group, and red arrows indicate neutrophils);

FIG. 3 is a kinetic analysis of His-pgp3 on conjugated antibody TNF-. alpha.;

FIG. 4 is a kinetic analysis of His-pgp3 on the conjugated antibody TNFR 1;

FIG. 5 shows the results of Hoechst33258 staining: a is TNF-alpha group, b is mixed group, c is blank group, d is pgp3 group;

FIG. 6 shows that Hoechst333258 detects apoptosis in each group of cells;

FIG. 7 is a flow chart of four sets of apoptosis (ordinate PI, abscissa Annexin V-FITC);

figure 8 is the value of interleukin 6 for each experimental group (×) P < 0.05: (a) statistical plots for the blank, pgp3 and TNF-. alpha.groups. (b) Statistical plots for the pgp3 group, TNF-. alpha.group and the mixed group. The horizontal axis of the two figures represents the group and the vertical axis represents the concentration of the corresponding IL-6.

Fig. 9 shows the values of interleukin 8 in each experimental group (×) P < 0.05, (a) is a statistical chart for the blank group, pgp3 group and TNF- α group, and (b) is a statistical chart for the pgp3 group, TNF- α group and mixed group. The horizontal axis of the two figures represents the group and the vertical axis represents the concentration of the corresponding IL-8.

Detailed Description

Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.

The invention will be described in detail with reference to the following examples.

The chlamydia protein pgp3 is prepared and purified by a prokaryotic expression technology, and is found to cause salpingitis reaction on a healthy mouse model, and can be specifically combined with TNF-alpha, so that the TNF-alpha induced apoptosis is effectively inhibited, and the chlamydia is ensured to complete the growth cycle in host cells. At the same time, both pgp3 and TNF-alpha induce an inflammatory response, stimulating the secretion of IL-6 and IL-8 by cells. pgp3 also synergistically enhances the intrafallopian inflammatory response by binding to TNF- α.

The sequence of the chlamydia plasmid encoded protein pgp3 is as follows:

(1) the protein is coded by Chlamydia trachomatis plasmid pORF5, has 795bp in total, and has the following nucleotide sequence (SEQ ID NO: 1):

(2) purified chlamydia protein pgp 3: the molecular weight is 29kDa, and the amino acid sequence is as follows (SEQ ID NO: 2):

the data in the following examples were analyzed using SPSS23.0 statistical software, and three replicates were performed for all experiments in this experimental study. Statistics are described using X + -S. If the data passes the homogeneity test of the variance, adopting a one-way ANOVA test; adopting rank sum test under the condition of uneven variance; p is less than 0.05, and the difference has statistical significance.

Example 1 construction of Experimental mouse model

1.1 preparation of laboratory animals

20 SPF-grade C3H/HeJ female mice with the body weight of 18-20g and the age of 5-6 weeks are bred.

1.2 Experimental groups and interventions

20 mice were randomly divided into 4 groups of 5 mice each, namely a blank group (without any treatment), a negative control group (namely, CT795 group), a high concentration group (His-pgp3 concentration of 10. mu.g/. mu.l), and a low concentration group (His-pgp3 concentration of 1. mu.g/. mu.l).

(1) The purified His-pgp3 was diluted to 10. mu.g/. mu.l and 1. mu.g/. mu.l with sterile PBS solution.

(2) His-pgp3 (10. mu.g/. mu.l, 10. mu.l) was injected into each of the left and right ovaries of the mice in the high concentration group, i.e., 100. mu.g of His-pgp3 was injected into each of the left and right ovaries of the mice.

(3) His-pgp3 (1. mu.g/. mu.l, 10. mu.l) was injected into each of the left and right ovaries of the mice in the low concentration group, i.e., 10. mu.g of His-pgp3 was injected into each of the left and right ovaries of the mice.

(4) The left and right ovaries of each mouse in the negative control group were injected with CT795 protein (10. mu.g/. mu.l, 10. mu.l), i.e., 100. mu.g CT795 was injected into each of the left and right ovaries of each mouse.

(5) All experimental mice were sutured immediately after intracyst injection. The mice were then housed in an SPF-grade animal house during which time temperature and humidity, food and water supplies were maintained. The mice were observed every other day for food intake, activity, incision status, presence of incision infection, etc. Note: the mouse intravesicular injection of the protein is carried out in a sterile environment, such as an ultra-clean bench operation.

1.3 Experimental animals were obtained and each group of mice was sacrificed at 14d by cervical dislocation, reproductive tract was isolated, photographed, and prepared into pathological section of oviduct tissue, and HE stained.

The oviduct specimen of the mice is shown in figure 1, the oviduct and the ovary of the mice in the blank group, the negative control group, the low-concentration group and the high-concentration group have no obvious swelling and hydrops, and the blood vessel of the oviduct wall has no dilatation and congestion.

HE stained sections of oviduct or ovary of each group of mice are shown in FIG. 2, in which the neutrophil cytoplasm is pink, the nucleus is large and bluish-purple, and the nuclear lobulation is evident. The number of neutrophils in each field was counted using image J software (see Table 1) and finally statistically analyzed using SPSS software. As can be seen from the experimental results shown in the following table, His-pgp3 stimulates the mouse oviduct to produce an inflammatory response, and is proportional to the concentration.

TABLE 1 neutrophils

The table shows the intrafallopian neutrophil counts of the mice, all values being expressed as mean ± sd. Comparing the high concentration group with the negative control group, wherein P is less than 0.001; comparison of # high concentration group and Low concentration group P < 0.001

Example 2 affinity assays for His-pgp3 and TNF-. alpha.and TNFR1

Diluted His-Pgp3 was immobilized on activated CM5 chips using amino acid coupling. TNF-. alpha.and TNFR1 were injected into the channels at different concentration gradients, and the signal intensity was measured. All signals are corrected via the blank channel. The analysis was performed using Biacore 3000Control Software.

Fig. 3 is a kinetic analysis graph of His-pgp3 on conjugated antibody TNF-alpha, fig. 4 is a kinetic analysis graph of His-pgp3 on conjugated antibody TNFR1, and it can be seen from fig. 3 and fig. 4 that as the concentration of TNF-alpha increases, the signal value increases and has a certain saturation trend, and the kd (m) value obtained by fitting is 2.05e-7 (fig. 3), but as the concentration of TNFR1 increases, the signal value linearly increases and has no saturation trend. His-Pgp3 and TNFR1 demonstrated non-specific binding (FIG. 4).

Example 3 apoptosis Rate detection

Detecting apoptosis rate of cells by Hoechest 33258

Removing the old culture medium in the 24-well plate, adding 1ml of PBS buffer solution into each well to clean the cells for 2-3 times, and removing the PBS buffer solution; adding 4% paraformaldehyde into each hole, and fixing for 15-20 min; washing with PBS buffer solution for 3 times, each time for 3-5 min; diluting the Hoechst33258 stock solution by 100 times by using normal saline or PBS buffer solution to obtain the working solution. Adding working solution into a 24-well plate, wherein 200 mu l of the working solution is added into each well, and dyeing is carried out at room temperature in a dark place for 3-5 min; removing the Hoechst33258 staining solution, and washing for 3 times with PBS (phosphate buffer solution) for 3-5 min each time; and observing the apoptosis condition under a fluorescence microscope.

The results of the experiment are shown in fig. 5, the mixed group has mostly uniform light blue (gray in fig. 5) and less nucleus-shrinkage compared to the positive control group; the cell nuclei morphology was more normal in the blank group.

2. Flow cytometry for detecting apoptosis rate

Washing the cells for 2-3 times by using a PBS buffer solution, and removing the PBS buffer solution; adding 500l pancreatin without EDTA into each well for digestion for 5min, and adding 1ml DMEM culture solution containing 10% FBS to stop digestion; transferring the cell suspension into a 1.5ml EP tube by using a pipettor, centrifuging at 1000rpm for 5 min; resuspending the cells with precooled PBS buffer, centrifuging at 1000rpm for 5min, and washing twice; preparing 1 × binding buffer, and adding 100l of binding buffer into each tube to resuspend cells; counting cells, adjusting the number of the cells to be 1 multiplied by 10^ 5/ml, and transferring 100l of cell suspension into a flow tube; respectively adding 5l of PE Annexin V and 5l of 7-AAD, and incubating for 15min at room temperature in the dark; adding 400l of 1 × binding buffer into each tube, and performing on-machine detection within 1 hour; note that: 2 tubes of single-stained cells (5 l PE Annexin V or 5l7-AAD was added) and 1 tube of double-negative cells (no dye was added) were prepared.

As shown in fig. 6 and 7, the apoptosis rate of the mixed group was decreased compared to the TNF- α group, i.e., the positive control group (p ═ 0.001, < 0.05); the pgp3 group had a reduced rate of apoptosis compared to the blank group (p ═ 0.036, < 0.05). The specific data are shown in Table 2.

TABLE 2 apoptosis of fallopian tube

Denotes the pgp3 group and the blank group P < 0.05, # denotes the mixed group and TNF-. alpha.group P < 0.05

From the test results of table 2, it can be seen that: the apoptosis rate of the mixed group was reduced by 26.3% compared to the positive control group (p ═ 0.001); the apoptosis rate of pgp3 group was 2.7% lower than that of the blank control group (P ═ 0.03, < 0.05).

3. Experiment of cellular inflammatory response

(1) The ELISA kit is placed in a normal temperature environment for balancing for 20min, the strips required for detection are taken out, and if the strips are redundant, the strips are packaged by a self-sealing bag and placed back in a refrigerator at 4 ℃.

(2) Blank holes, standard substances and samples are arranged, and 50 mul of standard substances with different concentrations are added into the holes of the standard substances according to the requirements of the specification. Adding 10 mul of sample to be detected into the sample hole interior line, and then adding 40 mul of sample diluent; blank wells were not added. Each hole is provided with 3-5 multiple holes.

(3) In addition to blank wells, 100. mu.l of detection antibody labeled with horseradish peroxidase (HRP) was added to both the standard and sample wells, the reaction wells were sealed with a sealing plate membrane, and incubated in a 37 ℃ water bath for 60min at constant temperature.

(4) After incubation is finished, removing liquid in the holes, beating on absorbent paper, filling washing liquid in each hole, standing for 1min, throwing off the washing liquid, and beating on the absorbent paper to be dry. This was repeated 5 times.

(5) 50. mu.l of substrate A, B was added to each well and incubated at 37 ℃ for 15min in the absence of light.

(6) The OD of each well was measured at a wavelength of 450nm within 15min by adding 50. mu.l of a stop solution to each well.

(7) Drawing a standard curve, calculating the concentration value of each sample according to a curve equation, and finally carrying out statistical analysis.

The values of interleukin 6 in the experimental groups are shown in FIG. 8, and it can be seen from FIG. 8 that pgp3 group can effectively stimulate IL-6 secretion from cells compared with blank control group; TNF-alpha group and blank group are compared, and cells can be stimulated to secrete IL-6; compared with the group of Pgp3 and TNF-. alpha.under the experimental conditions, His-Pgp3 is more capable of stimulating IL-6 secretion of cells. The pgp3 group was more effective in stimulating IL-6 secretion from cells than the mixed group; the mixed group stimulated IL-6 secretion from cells more than the TNF- α group. The above analysis is combined to show that: His-Pgp3 and TNF-alpha can stimulate oviduct cells to secrete IL-6, and after the His-Pgp3 and the TNF-alpha are combined, the stimulation effect is enhanced.

The values of interleukin 8 in the experimental groups are shown in FIG. 9, and it can be seen from FIG. 9 that pgp3 group is effective in stimulating IL-8 secretion from cells relative to the blank group; TNF-alpha group and blank group are compared, and cells can be stimulated to secrete IL-8; compared with the group of TNF-alpha, His-pgp3 was more able to stimulate IL-8 secretion from cells under the experimental conditions of pgp 3. The pgp3 group was more effective in stimulating IL-8 secretion from cells than the mixed group; the mixed group stimulated IL-8 secretion from cells more than the TNF- α group. The above analysis is combined to show that: His-Pgp3 and TNF-alpha can stimulate oviduct cells to secrete IL-8, and after the His-Pgp3 and the TNF-alpha are combined, the stimulation effect is enhanced.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the invention, so that any modifications, equivalents, improvements and the like, which are within the spirit and principle of the present invention, should be included in the scope of the present invention.

Claims

1. Use of chlamydia protein pgp3 in the manufacture of a medicament for the treatment of infertility in the fallopian tube.

2. Use according to claim 1, characterized in that: the fallopian tube infertility is infertility caused by salpingitis.

3. Use according to claim 2, characterized in that: the salpingitis is a tubal lesion caused by chlamydia protein pgp 3.

4. Use according to claim 2, characterized in that: the drug inhibits the development of salpingitis by inhibiting the binding of chlamydia protein Pgp3 to tumor necrosis factor TNF-alpha.

5. An inflammation inducing formulation characterized by: comprising the chlamydia protein Pgp3, said inflammation inducing agent inducing inflammation by inducing the chlamydia protein Pgp3 to bind to the tumor necrosis factor TNF- α.

6. The inflammation inducing agent according to claim 5, wherein: the inflammation is inflammatory lesion of oviduct induced by chlamydia protein pgp3, and stimulates oviduct epithelial cells to secrete IL-6 and IL-8.