Protein Kinase C Inhibitors For Candida Vaginitis Immune Deviation

PurposeCandida vaginitis, also known as vulvovaginal candidiasis (vulvovaginal candidiasis,VVC), is a common vulvovaginitis. Candida opportunistic pathogen has a total of ninepathogenic kinds to the human body, of the strongest and most common is Candidaalbicans[1]. It is reported the detection rate of Candida vulvitis in children vaginalsecretions is high, and mainly Candida albicans[2]. In recent years, the study of Candidaalbicans pathogenesis of recurrent vulvovaginal candidiasis, especially immunologicalmechanisms have some new progress. The study discoveries the immune balance betweenTh1and Th2cell abnormalities played a key role in[3]two recurrent pathogenesis ofvulvovaginal candidiasis. The study has showed that protein kinase C (PKC) plays acrucial role[4]in the response of Th2cells. The paper through inhibiting PKC----GF109203X inhibition of PKC activity and blocking the activation of T lymphocytes in themain pathway, changes the expression of the test animals, IFN-γ, IL-10and clarifies themechanism of the incidence of Candida vaginitis from the perspective of cell signaling.Through the study of animal models, this paper could provide theoretical basis for clinicalapplication PKC inhibitor and the treatment of the patients.Method1Experimental animals and groupingFeed the Kunming mice (sexually mature unmated, weight25g±5g) a week(excluding the environmental impact), after feeding mice a week, pick up four randomlyas blank control group (A), and the remained sixty mice are injected subcutaneously everyother day estradiol benzoate0.05ml/only (estradiol benzoate half-life of two-three days),a total of three times, so that the mice could in the false heat. Then pick purification of thestandard strain of Candida albicans ATCC10231on Sabouraud media. Use PBS to dilute the above bacteria liquid to7.5×106CFU/ml. Wash mice’s vaginal with salinerepeatedly，take twenty ul mixed bacteria fluid into mice and upside the mice for fiveminutes in order to ensure the bacteria liquid contact with the vagina fully and lasts sevendays，blank control group without any treatment，only raised with experimental mice at thesame time.Seven days later, observation experiment mice body, generally can be blankcontrol group mice, restless, hair density, smooth, bright color, eat better, depression ofinfected mice,the thinning hair and rough, matt color,eating less, it is observde theinfecting mice, the vaginal has more secretions, red and swollen. After cultivating thevaginal secretions, Candida grows, indicating that the infection was successful. Theinfected mice were randomly divided into a Group were immediately executed afterinfection group (group B) twelve (whichever successful after infection supernate save),infection without treatment group (group C) twelve, with vaginal infection medicationthree days group (D group) twelve, with vaginal infection medication three five group(E group) twelve, with vaginal infection medication seven days group (F group)twelve, prepare two-three more mice, a total of eighty-four.2Experimental MethodsAccording to table one take all mice’s ophthalmic vein blood after clotting naturally atroom temperature for after twenty minutes into the high-speed centrifuge (3000rev/min),collecting the supernatant on clear carried ELISA test line serum interleukin-10(Interleukin-10, IL-10), Interferon-γ(Interferon-γ, IFN-γ) changes in concentration,gather the mousse vaginal tissue specimens fixed in ten percent foer Ma Lin preserved toDali University affiliated Hospital pathology to carry on the paraffin embedding,0.5um forHE staining (hematoxylin-eosin staining); according to calculate the values of IFN-gamma, IL-10to calculate the ratio of IFN-gamma and IL-10represent the ratio of Th1and Th2to the mice, to understand the application of PKC inhibitors-after GF109203XTh1/Th2changes. Mice of group B line HE staining and PAS staining (observe mousevaginal cavity of hyphae and spores, further proof that mouse vaginal inflammation causedby candida albicans); Group A and group C eye venous blood and vaginal tissue time sameas the group F.3All kinds of calculation method 3.1The method of value about IL-10in serum、IFN–gamma and Th1/Th2According to specifications respectively measured by ELISA method in mice serumIL-10and the concentration of IFN-gamma, in a standard content as the abscissa, ODvalue as the ordinate, through draw the standard curve of the regression equation tocalculate the IL-10and IFN-gamma actual concentration; According to the actualconcentrations of IL-10, IFN-gamma to calculate the ratio of IFN-gamma and IL-10and the represented the ratio of Th1and Th2in mice.3.2Mouse vaginal infections criteria and scoring tableMouse vaginal inflammation criteria:0: mouse vaginal opening without congestion,edema and secretion;1: the vaginal opening mild hyperemia, edema in mice, no secretion;Two points: the mouse vaginal opening, swollen with secretion; Three points: the vaginalhyperemia, edema in mice, ulceration, accompanied by increased secretion; four points:death in mice.3.3Mouse vaginal tissue slicing criteria and scoring tableMouse vaginal tissue specimens according to congestion, edema, hemorrhage,infiltrate four evaluation index changes of mouse vaginal slices, each for one points.3.4Statistical MethodsSpss17.0experimental data are used for statistical analysis and processing software,all data were expressed as mean±standard deviation (x±s) indicated a significantdifference between the two tests of two independent samples using a paired t-test,correlation analysis linear correlation analysis, are ɑ=0.05level for the test, P <0.05considered statistically significant.Results(1) Group B, group C mice serum IL-10value higher than that of group A, IFN-gamma, Th1/Th2value lower than group A, the difference was statistically significant (P＜0.05), the mouse vaginal infection score and vaginal tissue section score were higherthan in group A.(2)Group D, E, F mice serum IL-10value higher than that of group A, IFN-gamma,Th1/Th2value lower than group A, the difference was statistically significant (P＜0.05).Mouse vaginal infection score and vaginal tissue section score were higher than in group A.(3) Group C mice serum IL-10value lower than group B, IFN-gamma value higherthan that of group B, the difference was statistically significant (P＜0.05), the Th1/Th2value higher than that of group B, there was no statistically significant difference. Mousevaginal infections a grade and vaginal tissue slice and group B with no difference.(4) Group D, E, F compared with group B and C in mice serum IL-10valuesgradually decline, IFN-gamma, Th1/Th2value increased gradually, the difference wasstatistically significant (P＜0.05). Group D, E, F mice vaginal infection score and vaginaltissue section scores were lower than that of group B, C.(5) Compared with group D mice group E IL-10in serum value is reduced, thedifference was statistically significant (P＜0.05), IFN-gamma, Th1/Th2value increases,there was no statistically significant difference. Group E mouse vaginal infection score andvaginal tissue section scores were lower than that of group D.(6)Group F compared with group D mice serum IL-10value decreased, IFN-gamma, Th1/Th2elevated (P＜0.05). Group F mouse vaginal infection score and vaginaltissue section scores were lower than that of group D.(7) Group F compared with group E IL-10in serum in mice value decreased, IFN-gamma, Th1/Th2elevated (P＜0.05). Group F mouse vaginal infection score and vaginaltissue section scores were lower than that of group E.Conclusions1.After candida vaginitis infection, infection in mice serum IL-10, IFN-gamma,Th1/Th2decreased (P＜0.05), the change of cell factors affect the occurrence of Th1/Th2immune deviation; Depression in mice, sparse hair, lacklustre, reduce food intake,vaginitis and vaginal tissue section high score.2.After vaginal application PKC inhibitors--GF109203X mice serum IL-10valuesgradually reduce, IFN-gamma, Th1/Th2values gradually rise, general improvement inmice, vaginitis and vaginal tissue section scores declined, suggests GF109203X may bereduced by vaginal yeast infection in mice serum IL-10values, IFN-gamma, Th1/Th2values, to play a regulatory role of Th1/Th2migration.3.Vaginal application PKC inhibitors-GF109203X for mice caused by Th1/Th2 immune deviation candida vaginitis has a certain therapeutic effect, and prolonged vaginaldrug for the treatment of candida vaginitis infection in mice is increased.