Effect Of Jieze No.1on The Innate Immune-related Factors In Defending Against Candida Albicans In Human Vaginal Epithelial Cells

Part One：Effect of JieZe NO.1on the Expression of TLR2and MyD88mRNA in Defending against Candida Albicans inCultured Human Vaginal Epithelial CellsObjective：To observe the effect of JieZe NO.1and the active component of it principal agent onthe natural immune factors（TLR2, MyD88）of human vaginal epithelial cells(VK2/E6E7); To demonstrate whether JieZe NO.1may play a role through TLRs signalpathway in human vaginal epithelial cells against C. albicans and clarify furthertheoretical basis of JieZe No.1on preventing and treatmenting with VulvovaginalCandidiasis.Methods:Set the blank control group (cultured VK2/E6E7alone), heat-killed C.albicansblastoconidia cultured together with VK2/E6E7(10:1per cent) for each time periodgroup (1h,3h,6h,12h,24h,48h). Extracted the total RNA of cells of each group, andthe expression of TLR2, MyD88mRNA was measured by real-time fluorescencequantitative PCR (QRT-PCR).Drug prevention experiments: set the blank control group, the model group(incubatedVK2/E6E7with C.albicans), cells+JieZe NO.1+C.albicans group, cells+Berberine+C.albicans group, and cells+Mannan+C.albicans group (incubated VK2/E6E7with drug, then plus C.albicans).Drug therapy experiments: set the blank control group, the model group (incubated theVK2/E6E7with C.albicans), cells+C.albicans+JieZe NO.1group, cells+C.albicans+Berberine group, cells+C.albicans+Mannan group (firstly co-cultured VK2/E6E7with C.albicans, then plus drug).The minimum non-cytotoxic drug concentration was determined by MTT. Aftercorresponding time, VK2/E6E7cells total RNA were extracted for TLR2, MyD88mRNA detection by real time quantitative PCR.Results:1. The Ct values of each group indicated that TLR2, MyD88mRNA was constitutivelyexpressed in the human vaginal epithelial cells (VK2/E6E7). After co-culturing withheat-killed C.albicans, the expression of TLR2, MyD88mRNA in VK2/E6E7was arising trend. In co-cultured12h, TLR2and MyD88mRNA were on highest expressionof amount, with significant difference in compare with negative control group (P<0.01).After24h, the expression declined, while the MyD88mRNA expression of24h incomparison with negative control group, still had a significant difference (P<0.05).2. The toxicity test of JieZe No.1, Berberine, Mannan on VK2/E6E7:MTT cytotoxicity assay drug: the IC10of JieZe NO.1on VK2/E6E7was14.7033mg/ml,time of24hours; the IC10of Berberine on VK2/E6E7was6.1645μmol/L, time of12hours; the IC10of Mannan on VK2/E6E7was24.1823mg/ml, time of2hours.3. The effect of JieZe NO.1, Berberine and Mannan on the expression of TLR2,MyD88of VK2/E6E7:Experiments of prevention: compared with the blank group, the expression of TLR2，MyD88mRNA about the cells in model group increased(P<0.01). Compared withmodel group, the expression of TLR2，MyD88in pretreated with JieZe NO.1orBerberine groups decreased significantly(P<0.01), and there were no obvious differencebetween the two groups. The expression of TLR2and MyD88was lower in the Mannan pretreatment group than unpretreated group (P<0.01),(P<0.05).Experiments of therapy: Compared with the model group, the expression of TLR2andMyD88in JieZe NO.1group increased (P<0.01),(P<0.05). the expression of TLR2andMyD88in the Berberine group increased (P<0.05), while the factors in Mannan grouphad no statistically significant changed(P>0.05).Conclusion:There were constitute a expression of TLR2and MyD88mRNA in immortalized humanvaginal epithelial cells（VK2/E6E7）. After co-culturing with heat-killed C.albicans,TLR2mRNA expression in VK2/E6E7increased clearly, with the expression ofMyD88was on a rising. Our findings suggest that C.albicans blastoconidia could berecognized by the TLR2on surface of VK2, may mediate innate immune responses viaMyD88-dependent pathway and start the downstream factors. TLR2may play a partialrole in the recognition of C.aibicans and startup host defense mechanism of C.albicansinfection of human vaginal epithelial cells. JieZe NO.1pretreated with cells can inhibitthe release of innate immune factors (TLR2、MyD88) effectively, and restrict the localinflammatory reaction; and for the cells which already infected with C.albicans, JieZeNO.1had further strengthen the innate immunity of cells in the early infected stages,and this function maybe implemented through TLR2-MyD88-dependent pathway. Part Two：Effect of JieZe NO.1on the Expression of HBD-2mRNA and IL-8, IL-1β in Defending against Candida Albicansin Cultured Human Vaginal Epithelial CellsObjective：To observe the effect of JieZe NO.1and the active component of it principal agent onthe inflammatory factors（HBD-2, IL-8, IL-1beta）of human vaginal epithelial cells(VK2/E6E7); To demonstrate whether JieZe NO.1may influence the secretion andexpression of infllammatory factors in human vaginal epithelial cells in defendingagainst C.albicans and clarify further theoretical basis of JieZe No.1on preventing andtreatmenting with Vulvovaginal Candidiasis.Methods：The prevention and therapy experiments of HBD-2: the same as the part one.The groups of IL-8and IL-1β: prevention experiments: set the blank control group, themodel group(incubated the VK2/E6E7with C.albicans), cells+JieZe NO.1, cells+Berberine (only treatmented the cells with drug), cells+JieZe NO.1+C.albicans group,cells+Berberine+C.albicans group(incubated the cells with drug, then plusC.albicans). Therapy experiments: set the blank control group, the model group(incubated the VK2/E6E7with C.albicans), cells+C.albicans+JieZe NO.1group, cells+C.albicans+Berberine group(firstly co-cultured the cells with C.albicans, then plusdrug).The method of experiments of IL-8and IL-1β: the cells incubated with the C.albicansor drug, then directly plus the appropriate concentration of drug or C.albicans. Afterco-culturing, Supernatant from VK2/E6E7cells were collected for cytokines(IL-8,IL-1β) analysis by ELISA.Results: 1. the effect of JieZe NO.1, Berberine and Mannan on the expression of HBD-2mRNAof VK2/E6E7: compared with the blank group, the expression of HBD-2mRNA in themodel group increased(P<0.01). Compared with model group, the expression of HBD-2in pretreated with JieZe NO.1, Berberine and Mannan groups decreasedsignificantly(P<0.01). Compared with the model group, the therapy group of JieZeNO.1increased (P<0.05), while the expression of HBD-2in Berberine and Mannandecreased significantly(P<0.01).2. the effect of JieZe NO.1and Berberine on inflammatory factors（IL-8, IL-1beta）ofVK2/E6E7:Experiments of prevention: compared with the blank group, the expression of IL-8in model group decreased significantly(P<0.01), the expression of IL-8in JieZe NO.1group had no statistically significant change, but the Berberine group decreasedsignificantly(P<0.01). Compared with the model group, the expression of IL-8in JieZeNO.1and Berberine prevention groups increased(P<0.01). Compared with the JieZeNo.1group, the IL-8in the JieZe NO.1pretreated group was lower(P<0.01). The IL-8in the Berberine pretreated group was higher than the Berberine group(P <0.01). Theexpression of IL-1β had no significant change among all the groups.Experiments of therapy: compared with the model group, the expression of IL-8inJieZe NO.1therapy group had no significant change. But the Berberine therapy groupdecreased in the expression of IL-8(P<0.01). The expression of IL-1β had nosignificant change among the groups.Conclusion:JieZe NO.1can significantly inhibit the expression of HBD-2mRNA in preventinginfection, and this maybe limit local inflammatoy. With the cells had been infected withC.albicans, JieZe NO.1can increased the expression of HBD-2, which play a role inkilling C.albicans.The human vaginal epithelial cells (VK2/E6E7) spontaneously released IL-8and IL-1β. After co-incubated with heat-killed C.albicans blastoconidia, the ability of releaseingIL-8in VK2reduced, this maybe the one of causes of pathopoiesis. It had no effect onthe secretion of IL-1β. JieZe NO.1did not change the ability of releaseing IL-8in VK2,and use JieZe NO.1and Berberine as prophylactic, could improve the ability of cells ofreleasing IL-8when infected. JieZe NO.1had no significant effect on the release ofIL-1β in VK2.