| Objective1.To explore whether the antifungal traditional Chinese medicine may defend the vaginal candidiasis of mice.2.To explore effects of traditional Chinese medicin in mice by the differents expressions of TLR2,TLR4,IL-6,IL-10,IL-12.This eploration offers new methods and thinkings for further experiment.Methods1.To collect the ancient records by philological method and to establish database after data standardization treatment.Researches of medical frequency,clustering and association rules were analyzed by zhongyichuancheng software platform after data entry.Unearthing the intrinsic relationship between medicine and medicine in daixia disease and summarizing the similarity between daixia disease and vulvovaginal candidiasis through data analysis.The treatment medical rule of vulvovaginal candidiasis was got from the basis of data analysis.2.The number of 65 female BALB/c mice(8 weeks)with weight of 20 to 25g were randomly divided into 3 groups including Chinese traditional medicine group and model group,blank group.There were 30 mice in each traditional Chinese medicine group and model group,while there were only 5 mice in blank group.The model group mice were intragastric administration with saline since the first day and were subcutaneously injected 0.1 mL containing 0.1 mg pentanoic acid estradiol in the 4th day,then took each injection once a week.In the seventh day,the model group mice vagina inoculated with 100μl C.albicans 14053 suspensions containing spores in the quantity of 10~8 bacteria.The mice in Chinese traditional medicine group were intragastric administration with antifungal traditional Chinese medicine since the first day and were subcutaneously injected 0.1 mL containing 0.1 mg pentanoic acid estradiol in the 4th day,then took each injection once a week.In the seventh day,the Chinese medicine group and model group mice vagina inoculated with100μl C.albicans 14053 suspensions containing spores in the quantity of 10~8bacteria.The blank group mice were intragastric administration with saline since the first day and were subcutaneously injected 0.1 mL containing 0.1mg pentanoic acid estradiol in the 4th day.3.In the 8th d,9th d,10th d,12th d and 14th d and 21th d(namely,C.albicans infection after 1st d,2nd d,3rd d,5th d,7th d,14th d),traditional Chinese medicine group and model group randomly selected five mice in each group.These mice were put to execution and then collected vaginal lavage and vaginal tissue after anesthesia.The 5 mice in blank control group were all put to execution and then collected vaginal lavage and vaginal tissue after anesthesia in the 8th day.4.These mice were taken for vaginal douche with 0.1 ml containing 100units protease inhibitors(complete,EDTA free)sterile PBS buffer after anesthesia.Vaginal lavage 40μl diluted in 10-1000 times,100μl dilution were cultivated with spread plate method on the yeast chloramphenicol glucose agar culture medium under 37℃in 24 h.The number of colony was counted after observing the tablet colony growth situation.5.At the same time,vaginal lavage diluted in proper multiples,to test the change of the IL-6,IL–10,IL–12 in mice vaginal lavage using ELISA kits.6.Vaginal tissue in each mice was divided into two parts,one tested TLR2and TLR4 expression levels by real-time fluorescent quantitative PCR detection.The other part was made of wax blocks to detect expression levels of TLR2 and TLR4 protein in the vaginal tissue changes of mice by immunohistochemical staining.Results1.Colony count:CFU in model group mice vaginal lavage transient rised,and then gradually declined after peaking in the third day,which maintained at a low level.The CFU changing trend of mouse vaginal lavage in Chinese traditional medicine group was not increased obviously during the first three days,and the CFU of mouse vaginal lavage in Chinese traditional medicine group was obviously lower than that of model group.In the 3rd day,5th day,7th day after infection,two groups of mice vaginal lavage CFU was in obvious difference(P<0.05).Traditional Chinese medicine mice appeared negative with one in the 3rd day after infection and two in the 7th day after infection.C.albicans wrer not found in blank control group mice vaginal lavage.2.RT-PCR and immunohistochemical staining:TLR2 and TLR4 gene expression were all increased in model group and traditional Chinese medicine group.Model group expression of TLR2 grew stablely in a range while TLR4 expression transient rised reaching peak in the 7th day after infection,and then began to decline.In Chinese traditional medicine group the addition of TLR2expression was stable in a range,but the growth rate was significantly faster than the model group.The two groups had significant differences(P<0.05)in the 2nd till the 14th day.The expression trend of TLR4 in Chinese traditional medicine group reached a peak in the 3rd day after infection,which was the same with the model group.The two groups had significant differences(P<0.05)in the 3rd,5th,7th day.The growth rate of TLR4 gene was greater than the model group.The result of immunohistochemical found that TLR2 and TLR4had the same change trend with real-time quantitative PCR results.Expression quantity of protein in traditional Chinese medicine group was greater than the model group.TLR2 had significant differences in model group and Chinese traditional medicine group(P<0.05)in the 1st,3rd,5th,7th day.TLR4 had significant differences in model group and Chinese traditional medicine group(P<0.05)in the 1st,3rd,5th,7th,14th day.The expression of TLR2 and TLR4had a positive correlation with immunohistochemical results(P<0.05).3.Cytokines:IL–6 and IL-12 in mouse vaginal lavage increased compared with the blank group,while IL-10 was falled.IL–6 had a continously growing in vaginal lavage of model group and Chinese medicine group.But there was no obvious difference between model group and traditional Chinese medicine group(P>0.05).IL-10 in vaginal lavage declined in model group and traditional Chinese medicine group compared with blank control group.The level of IL–10 fallen within a low level,which had significant difference between the two groups(P<0.05)in the 1st and 2nd day after infected.In model group IL-12 increased significantly in the 3rd day after infection and declined in the 7th day after infection.In Chinese traditional medicine group,IL–12 increased occurred in the 3rd day and then declined,but the level of IL-12 is lower than the model group.In the 3rd day,5th day,7th day after infection,IL-12 had significant differences in model group and Chinese traditional medicine group(P<0.05).The expression of TLR2 mRNA had a negative correlation with the concentration of IL-10(P<0.05)in the vaginal enviorenment.Conclution1.The antifungal traditional Chinese medicine could partly preventing infection of C.albicans,which could significantly reduce the number of c.albicans in the vagina.2.Mice could defense C.albicans infection through TLR2 and TLR4regulation.The infection of C.albicans in mice mucusa may relate with the unbalance of IL-6,IL-10,IL-12.3.Antifungal traditional Chinese medicine intervents infection of C.albicans through the TLR2 and TLR4 signaling pathways.The antifungal traditional Chinese medicine may militate through influencing the expression of IL-10 and IL-12.4.The up-regulation through the TLR2 signaling pathways inhibite the expression of IL-10,which may play an important role in the mice mucusa infection of C.albicans. |
