| Objective:To investigate the anti-infective effect of peptidoglycan recognition protein 1(PGLYRP-1)in reproductive tract model of chlamydia muridarum(Cm),and explore the protective efficacy and protection mechanism against Cm infection in female BALB/c mice.Methods:The PGLYRP-1 gene was amplificated by PCR.The prokaryotic expression plasmid of pET-28a(+)/PGLYRP-1 was constructed and transformed into E.coli BL21.The recombinant protein was induced by IPTG;and identified by western blot.The concentration of PGLYRP-1 was determined by the BCA method,and the polymyxin B was used to remove the endotoxin of the recombinant protein.Mice with 6-8 weeks old were injected with dope-provera 2.5 mg.After 1 week,they were divided into three groups.One group was a PBS control,the same volume of PBS was injected.One group was infected with Cm as a positive controlvia mice posterior vaginal fornix,the injected numbers of Cm inclusion bodies were5.0×10~5 IFUs,15μL of each mice,and a model of genital tract infection was established.One group was an anti-infective group of PGLYRP-1 protein,which was divided into three different doses,50μg、100μg、200μg,and each dose was inject into mice posterior vaginal fornix.After the Infection of Cm,the genitalsecretions were collected with cotton swabs on 3d、7d、10d、14d、21d、28d、35d and the number of inclusion bodies was determined by IFA method.On the 21st day of infection,the mice were sacrificed by cervical dislocation,and the genital tract(vaginal,uterus,fallopian tube,ovary)of the mice were aseptically taken.The expression level of IL-2、IL-4、IFN-γcytokines in the oviduct homogenate were measured by ELISA.After the 63day of infection,the genital tissues of the mice were aseptically isolated,and the oviduct sections were stained with hematoxylin-eosin(H&E)to evaluate the pathological changes.Results:1.After induction of PGLYRP-1 recombinant protein,the purpose appears at about 24 kDa protein banding.2.After 3d、7d、10d、14d、21d、28d、35d of infection,the genitalsecretions werecollected to calculate the numbers of inclusions.The numbers of inclusions in positive group were(10.8±0.80)×10~4、(1.0±0.96)×10~4、(1.2±1.00)×10~3、(4.0±0.50)×10~2、(1.2±0.25)×10~2、(4.0±0.08)×10、there is no detected inclusion in 35day.The numbers of inclusions in50μg PGLYRP-1 anti-effective group were(9.8±0.72)×10~4、(8.0±0.82)×10~3、(5.0±0.24)×10~2、(4.0±0.80)×10、there is no detected inclusion after 21day.The numbers of inclusions in 100μg PGLYRP-1 anti-effective group were(9.6±0.84)×10~4、(2.5±0.60)×10~3、(2.2±0.64)×10~2、there is no detected inclusion after 14day.The numbers of inclusions in 200μg PGLYRP-1 anti-effective group were(8.8±0.49)×10~4、(4.2±0.88)×10~2、there is no detected inclusion after 10day.Compared to control group,the injection of PGLYRP-1 can accelerate the clearance of Cm in genital tract of mice.3.The mice were sacrificed on the 63day and the mice were examined for fallopian tube lesions.There were 5 of the 6(5/6)patients in the Cm infection group have shown unilateral or bilateral water accumulation,and the lesion rate reached 83.33%,while PGLYRP-1 anti-infective group 2 of the 9(2/9)have shown unilateral or bilateral water accumulation,the morbidity is only 22.22%.In the prepared H&E sections,when the microscope was magnified 40 times,inflammation was counted on both side walls of the mice and the oviduct lumen.The PGLYRP-1 protein anti-infective group was significantly different from the Cm-positive group according to the inflammatory cell infiltration score(P<0.05).Compared with the macroscopic between anti-infective group and the Cm-positive group,the fallopian tube dilatation scores were statistically different(P<0.05),while the microscopic tubal dilatation score was consistent with the results of hydrops.These data indicate that the oviduct tissue in the positive control group posses an obvious intraluminal irregular expansion,water accumulation and inflammatory cell infiltration.Compared with the positive control group,the degree of hydrosalpinx and inflammation in oviduct tissue in PGLYRP-1 anti-infected group were clearly relieved,and the infiltration of inflammatory cells was reduced,The degree of reduction is directly proportional to the PGLYRP-1 protein content.4.The supernatant of tubal tissue was collected in 21day after Cm infectionto determine the levels of cytokines IL-2、IL-4、IFN-γ.The IL-2was not statistically available.The expression of IL-2 was 8.24±0.15pg/mL in PBS control group,8.52±1.29pg/mL in positive control group,and8.68±0.67pg/mL、9.03±0.38pg/mL、9.44±0.76pg/mL in goups with different concentration of PGLYRP-1.The difference was no significant(P>0.05).The expression of IL-4 was 2.21±0.50pg/mL in PBS control group,12.42±0.86 pg/mL in positive control group,and 11.73±1.38 pg/mL、15.56±0.92pg/mL、20.00±1.84pg/mL in the goups with different concentration of PGLYRP-1.Positive group and anti-infective group both were significantly higher than the PBS control group(P<0.05),and the expressions of IL-4 in 100μg and 200μg anti-infective group were higher than the positive group(P<0.05).The expression of IFN-γwas 14.02±2.33pg/mL in PBS control group,89.77±3.96 pg/mL in positive control group,and 65.41±2.54 pg/mL、117.57±8.55 pg/mL、143.23±9.67 pg/mL in goups with different concentration of PGLYRP-1.The positive group and anti-infective group both were significantly higher than the PBS control group(P<0.05),and the expressions of IFN-γin 100μg and 200μg anti-infective group were higher than the positive group(P<0.05).The degree of reduction is directly proportional to the PGLYRP-1 protein content.Conclusion:PGLYRP-1 made an anti-effective in gentital tract of mice infected with chlamydia muridarum. |
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