| Objective:This study for chlamydia genital tract infection in mice model,kang fu yan capsules on effect on the improvement of the genital tract inflammation and fertility,the immunomodulation mechanism of kang fu yan capsule in the genital tract after CT infection.Materials and methods:1.170 female ICR mice were randomly divided into 2 groups:Blank control Group(10),Chlamydia infection mice(genital tract inflammation Model control group)(160).The model mice were randomly divided into 5 groups,namely:Model control Group(30),Kang Fu Yan with low dosage Group(30),Kang Fu Yan with medium dosage Group(30),Kang Fu Yan with High dosage Group(30),azithromycin Group(30).In the low,middle and high dosage group,the mice were given 0.154g/kg,0.46g/kg,1.404g/kg dosage of Kang Fu Yan,the drug volume of 10ml/kg,1 time a day.Azithromycin group of mice were given 0.13g/kg of azithromycin,dosing volume for 10ml/kg,once.The normal group and model group of mice were given the same volume of double steamed water.In each group,10 mice were randomly selected from 1w,3w and 5w to pick the blood anticoagulant from eyeball,anatomical observation of morphological changes of genital tract,HE staining to observe the infiltration of inflammatory cells.expression of Cdllb in immunohistochemical assay,detection of the content of uterine myelin peroxidase(MPO)by ELISA and detection of mouse reproductive ability.2.The neutrophils were isolated from the rat and cultured in vitro in the six-orifice plate after cell adherent to give a certain dose of chlamydia infection(chlamydia infection dose is mio=2,infection time is 6h),and Kang Fu Yan drug treatment(Kang Fu Yan drug concentration is 10ng/10?g/lmg,medication time is 6/12/24 hours).Detection of the levels of ROS,MPO in neutrophils after treatment of chlamydia infection with Kang Fu Yan,Changes in the concentration of chlamydia in medium in the post-treatment medium,the effects of TLR2/4 activation and inhibitors on the activity of kang fu yan capsule regulating neutrophils.Correlation molecular mechanism of regulating neutrophil activity by TLRS-related receptor activator and inhibitor.Results:1.The results of animal experiment(1)Pathologic Anatomy of the genital tract:Chlamydia infected mice vagina 5 weeks later,can cause vaginal mouth redness,the uterus swelling and water performance,HE dyes the inflammatory cells infiltrating.Kang Fu Yan capsule treatment 1 week?3 weeks and 5weeks later,found to reduce chlamydia infection caused by swelling and water in the uterus of mice,HE staining showed slight improvement in inflammatory cell infiltration in mice uterus.(2)Immunohistochemical detection of the expression of uterine Cdllb in mice:The expression levels of CDllb in mice with chlamydia genital tract infection were significantly higher than that in the blank group,and there were significant differences after the 1w,3w and 5w(P<0.05).Compared with the expression level of CD11b in the model group,azithromycin group decreased significantly(P<0.05),and the expression level of CD11b in the Kang Fu Yan group was reduced to a certain extent compared to the model group,the difference was statistically significant after 5weeks(P<0.05).(3)Detection of the content of uterine myelin peroxidase(MPO)by ELISA:Chlamydia genital tract infection of mice model of uterus tissue MPO expression level was significantly higher than that of blank group,azithromycin group and kang fu yan capsule in the degree of concentration and high concentration group were lower than that of model group,high concentrations of kang fu yan group compared with model group have significant difference(P<0.05),low concentration kang fu yan group there was no significant difference.(4)Detection of reproductive ability in mice:In the blank group,the mice had the shortest reproductive time,the model group showed an increase(P<0.01),Compared with the model group,the time of fertility was reduced in the kang fu yan group,but there was no significant difference.two mice unconceived in Sixteen mice in the model group(9w),12.5 percent infertility rates,and all 16 mice in the kangfu group were pregnant.Kang fu yan group had a significantly higher number of foal than model group(P<0.05).A group of female rats were dead in both the kang fu yan group and the model group,and four baby mice were dead in the model group.2.The results of cell experimental(1)Reducing the production of ROS by Kang Fu Yan:Chlamydial infection after neutrophils,intracellular ROS level increased significantly(P<0.001),while kang fu phlogistic capsule after processing cells significantly inhibit the generation of ROS in cells,including three time points of K 1mg,and K 10?g and 10 ng·ml-1 of 12 h and 24 h compared with chlamydia group had significant difference(P<0.001).(2)Reducing the production of MPO by Kang Fu Yan:After chlamydia infected neutrophils,the secretion of MPO increased,but no significant difference was found.In kang fu yan capsule treatment cell,cell cultures of MPO levels are suppressed,in which three time points of K-1mg and K-10?g and 10 ng ·ml-1 of 12 h and 24 h compared with chlamydia group had significant difference(P<0.05).(3)Kang fu yan capsule promotes neutrophil phagocytosis chlamydia:kang fu yan capsule after processing,the detection of chlamydia charge cell cultures of loads,found kang fu yan capsule significantly reduced concentration of medium,Chlamydia(P<0.01).(4)The effect of TLR2/4 activation and inhibitor on the regulation of neutrophils in kang fu yan capsule:the capsule significantly reduced the amount of chlamydia in the medium(P<0.05).When TLR2 agonist PGN(10?g · ml-1)and TLR4 agonist LPS(100ng·ml-1)were respectively applied to 6h,the load of chlamydia was significantly lower than that of the kangfu group(P<0.01,P<0.001).In contrast,after the TLR2 antibody and TLR4 antibody 10?g·ml-1)were respectively applied to 6h,the load of chlamydia was basically the same as that of the kang fu yan group.For the secretion of MPO,TLR2/4 agonist effect after there was no significant difference with the kang fu yan group,whereas its inhibitor effect after compared with kang fu yan group decreases,which TLR4 antibody group with significant difference(P<0.05).Conclusion:Kang fu Yan capsule is used in the treatment of chlamydia infection,promote PMN phagocytosis,regulate PMN sterilization,by reducing the activity of neutrophils to a certain extent,Inhibit PMN generation and secretion of ROS and MPO,and reduce tissue damage caused by PMN degranulation release ROS and MPO.Thus reducing the normal tissue,cell damage,improve fertility,But its regulation on molecular mechanism of PMN sterilization ways do not overlap with TLR2/4 signaling pathways. |
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