| Vulvovaginal candidiasis(VVC),also known as Candida vaginitis,is a mucosal inflammatory disease mainly induced by Candida,which causes problems in the life of the majority of women.The treatment of VVC with antifungal chemical drugs faces severe challenges,such as high adverse reaction rate,obvious side effects,prone to relapse,and the emergence of drug-resistant and cross-resistant strains.The development of a new generation of antifungal drugs is slow,and there is a long way to find new antifungal target drugs.There is rich clinical experience in the external therapy of VVC with traditional Chinese medicine,and the curative effect is definite.It has the characteristics of low side effects,low induced drug resistance,low price,suitable for long-term and preventive use.Its finished medicine has good clinical value and market prospects.ObjectiveIn recent years,the research group has carried out continuous research on Kangbainian(KBN)lotion,a clinical prescription for treating VVC,based on the research orientation of clinical prescriptions.KBN lotion is confirmed that it has a good anti-fungal effect on sensitive Candida albicans(C.albicans),and has a good medicinal effect on VVC mice colonized with sensitive C.albicans.Whether KBN lotion affects drug-resistant C.albicans and VVC model mice induced by them,and the mechanism needs to be further studied.Therefore,this study simulates an in vitro-in vivo experiment to explore the above scientific issues and provide a scientific experimental basis for KBN lotion in the treatment of refractory VVC.Methods1 Effect and mechanism of KBN lotion against drug-resistant C.albicans in vitro1.1 Study on the anti-fungal activity of KBN lotion on drug-resistant C.albicansThe minimum inhibitory concentration(MIC)of KBN lotion on sensitive and drugresistant C.albicans was determined by in vitro anti-fungal susceptibility test.The antifungal activity of KBN lotion on sensitive and drug-resistant C.albicans was dynamically monitored by a time-killing curve test.The relationship between concentrations of KBN lotion and sterilization time of sensitive and drug-resistant C.albicans was investigated through the sterilization time experiment.1.2 Effect of KBN lotion on biofilm,hyphae,and adhesive of drug-resistant C.albicansThe effect of KBN lotion on the early biofilm formation and mature biofilm of sensitive and drug-resistant C.albicans was measured by the XTT experiment.The effect of KBN lotion on the hyphae growth of sensitive and drug-resistant C.albicans were observed by hyphae growth experiment.The effect of KBN lotion on sensitive and drug-resistant C.albicans adhering to non-living surfaces,human vaginal epithelial cells(VK2/E6E7),and human cervical cancer cells(Hela)were observed under fluorescence microscopy by calcofluor white stain(CFW).1.3 Effect of KBN lotion on the ergosterol synthesis and the expression levels of related gene ERG11 in drug-resistant C.albicansThe effect of KBN lotion on the ergosterol synthesis of drug-resistant C.albicans was detected by high-performance liquid chromatography(HPLC).The effect of KBN lotion on the expression level of ERG11 gene in drug-resistant C.albicans was detected by reverse transcription-polymerase chain reaction(RT-PCR).1.4 Effect of KBN lotion on the function and gene expression levels of drug-resistant C.albicans drug transporterThe effect of KBN lotion on the drug transporter absorption and efflux function of drugresistant C.albicans by Rhodamine 6G/123(Rh 6G/123)absorption and efflux experiment.The effect of KBN lotion on the expression levels of drug-resistant C.albicans efflux pump genes(CDR1,CDR2,and MDR1)was detected by RT-PCR.2 Study on the effect and mechanism of KBN lotion on VVC model animals caused by drug-resistant C.albicans in vivo2.1 Effect of KBN lotion on VVC model mice caused by drug-resistant C.albicansVVC mice model was established by subcutaneous injection with estrogen and vaginal inoculation with drug-resistant C.albicans 901,and 30 μL of corresponding drugs were administered vaginally to the mice for 3 weeks.The pharmacodynamic indicators include the symptoms of mice vaginal inflammation,the morphology of drug-resistant C.albicans in the vaginal lavage fluid was observed by gram staining,the load of drug-resistant C.albicans in the vaginal lavage fluid was measured by plate counting,the histopathological change in the vagina was observed by HE staining,PAS staining,and scanning electron microscope.2.2 Effect of KBN lotion on VVC model rat caused by drug-resistant C.albicansVVC rat model was established by subcutaneous injection with estrogen and vaginal inoculation with drug-resistant C.albicans 901,and 150 μL of corresponding drugs were administered vaginally to the rat for 1 week.The pharmacodynamic indicators were the same as those of the drug-resistant VVC model mice.2.3 Anti-inflammatory mechanism of KBN lotion on VVC model mice caused by drugresistant C.albicansThe expression levels of mice vaginal tissue inflammatory factors(TNF-α,IL-1 β,and IL-6),inflammatory body NLRP3,and dendritic cell-associated C-type lectin-1(Dectin-1)signaling pathways(Dectin-1,Syk,PLCy-2,CARD9,and NF-κB)protein were detected by western blotting(WB).3 The pharmacodynamic substances and potential advantages of KBN lotion against drug-resistant C.albicans3.1 The pharmacodynamic substances of KBN lotion against drug-resistant C.albicansThe MIC of the monomeric component in KBN lotion against drug-resistant C.albicans was detected by in vitro anti-fungal susceptibility tests.3.2 Study on the combined anti-fungal effect of KBN lotion and azoles on drug-resistant C.albicansThe combined anti-fungal effect of KBN lotion and azoles on drug-resistant C.albicans was evaluated by the fractional inhibitory concentration index(FICI)using the checkerboard micro liquid-based dilution method.3.3 Study on the combined anti-fungal effect of monomer components in KBN lotion or monomer components/azoles on drug-resistant C.albicansThe combined anti-fungal effect between some monomer components in KBN lotions or between monomer components/azoles drugs on drug-resistant C.albicans was evaluated by the FICI using the checkerboard micro liquid-based dilution method.3.4 Study on KBN lotion reversed drug-resistant C.albicans resistance to fluconazoleThrough stimulation and induction by KBN lotion,it was investigated whether KBN lotion could restore drug-resistant C.albicans sensitivity to fluconazole and whether the progeny of drug-resistant C.albicans were resistant to KBN lotion.Results1 Effect and mechanism of KBN lotion against drug-resistant C.albicans in vitro1.1 Study on the anti-fungal activity of KBN lotion on drug-resistant C.albicansThe results showed that drug-resistant C.albicans(901,904,311)were resistant to fluconazole(MIC>64 μg/mL)and ketoconazole(MIC>8 μg/mL)through in vitro anti-fungal susceptibility tests.KBN lotion has the same anti-fungal effect on drug-resistant C.albicans,and its MIC was 6.25 mg/mL which was lower than that of Xianglian waixiye and Jieeryin xiye.The results showed that KBN lotion inhibited the growth of sensitive and drug-resistant C.albicans,and had a better anti-fungal effect with the higher concentration by the time-kill curve experiment.The inhibitory effect of 12.5 mg/mL KBN lotion on sensitive and drugresistant C albicans was better than that of Jieeryin xiye with the same dilution ratio of original solution.The results showed that KBN lotion sterilized sensitive and drug-resistant C.albicans by the sterilization time experiment.The higher concentration,the shorter time of the sterilization.The time of 1.00 g/mL KBN lotion sterilized sensitive and drug-resistant C.albicans(15 min)was shorter than the original Jieeryin xiye(360 min).1.2 Effect of KBN lotion on biofilm,hyphae,and adhesive of drug-resistant C.albicansThe results showed that KBN lotion effectively inhibited the formation of the early biofilm and destroy the mature biofilm of sensitive and drug-resistant C.albicans by XTT experiment.The results showed that KBN lotion inhibited the growth of sensitive and drugresistant C.albicans hyphae by the hyphae growth experiment.The results showed that KBN lotion inhibited the adhesion of sensitive and drug-resistant C.albicans to abiotic surfaces,VK2/E6E7 cells,and Hela cells,inhibited the growth of hyphae,and protected VK2/E6E7 cells to a certain extent by adhesion experiments.1.3 Effect of KBN lotion on the ergosterol synthesis and the expression levels of related gene ERG11 in drug-resistant C.albicansThe results showed that KBN lotion significantly reduced the ergosterol synthesis of drug-resistant C.albicans 901 by HPLC experiment.The results showed that KBN lotion significantly down-regulated the expression of ERG11 gene by RT-PCR experiments.1.4 Effect of KBN lotion on the function and gene expression levels of drug-resistant C.albicans drug transporterThe results of Rh 6G/123 absorption experiment showed that KBN lotion significantly promoted the absorption of Rh 6G/123 in drug-resistant C.albicans 901.The results of Rh 6G/123 efflux experiment showed that the efflux of Rh 6G/123 by drug-resistant C.albicans 901 was significantly higher than that of sensitive C.albicans 98001,and KBN lotion significantly inhibited the efflux of Rh 6G/123 in drug-resistant C.albicans 901.The results of RT-PCR experiment showed that the expression of efflux pump genes(CDR1,CDR2,and MDR1)in drug-resistant C.albicans 901 was significantly higher than that in sensitive C.albicans 98001,and KBN lotion significantly inhibited the overexpression of efflux pump genes(CDR2 and MDR1)in drug-resistant C.albicans 901.2 Study on the effect and mechanism of KBN lotion on VVC model animals caused by drug-resistant C.albicans in vivo2.1 Effect of KBN lotion on VVC model mice caused by drug-resistant C.albicansThe experimental results of VVC model mice showed that KBN lotion significantly improved the symptoms of vaginal redness and swelling,reduced the amount of white secretion and fishy odor,significantly reduced the vaginal drug-resistant C.albicans load,and inhibited the hyphal formation of VVC model mice.The results showed that KBN lotion improved the degree of vaginal inflammation and inhibited the growth and hyphal formation of drug-resistant C.albicans in the vagina of VVC model mice by the HE and PAS staining.The results showed that KBN lotion restored the smoothness of the inner surface of the vaginal tissue of VVC model mice,inhibited drug-resistant C.albicans adhesion and hyphal formation by scanning the vaginal tissue using electron microscopy.The medicinal effect of KBN lotion on VVC model mice was better than the marketed drug Jieeryin xiye.2.2 Effect of KBN lotion on VVC model rat caused by drug-resistant C.albicansThe experimental results of VVC model rat showed that KBN lotion significantly improved the symptoms of vaginal redness and swelling,reduced the amount of white secretion,significantly reduced the vaginal drug-resistant C.albicans load,and inhibited the hyphal formation of the VVC model rat.The results showed that KBN lotion improved the degree of vaginal inflammation,inhibited the growth and hyphal formation of drug-resistant C.albicans in the vagina of VVC model rat by the HE and PAS staining.The results showed that KBN lotion inhibited drug-resistant C.albicans adhesion and hyphal formation in the vagina of VVC model rat by scanning vaginal tissue using electron microscopy.The medicinal effect of KBN lotion on VVC model rat was better than the marketed drug Jieeryin xiye.2.3 Anti-inflammatory mechanism of KBN lotion on VVC model mice caused by drugresistant C.albicansThe results of WB experiment showed that the protein expression levels of TNF-α,IL1β,and IL-6 in the vaginal tissue of the drug-resistance VVC model group were significantly increased than that of the control group,the protein expression level of NLRP3 was significantly increased than that of the control group,and the protein expression levels of Dectin-1,Syk,PLCy2,Card9,and NF-κB were significantly increased than that of the control group.The expression levels of the above proteins in the KBN lotion group were significantly decreased than that of the model group.3 The pharmacodynamic substances and potential advantages of KBN lotion against drug-resistant C.albicans3.1 The pharmacodynamic substances of KBN lotion against drug-resistant C.albicansThe results of in vitro anti-fungal susceptibility test showed that berberine(MIC 32μg/mL),palmatine(MIC 128 μg/mL),coptisine(MIC 128 μg/mL),thymol(MIC 320μg/mL),carvacrol(MIC 320 μg/mL),and eugenol(MIC 1280 μg/mL)had the significant anti-fungal effect on sensitive C.albicans 98001 and drug-resistant C.albicans(901,904,311).3.2 Study on the combined anti-fungal effect of KBN lotion and azoles on drug-resistant C.albicansThe results of checkerboard micro liquid-based dilution method showed that KBN lotion/fluconazole had a synergistic effect against sensitive C.albicans 98001 and drugresistant C.albicans 901(FICI respectively:0.28,<0.09),KBN lotion/miconazole nitrate and KBN lotion/ketoconazole had a synergistic effect against drug-resistant C.albicans 901(FICI respectively:0.31,<0.16)3.3 Study on the combined anti-fungal effect of monomer components in KBN lotion or monomer components/azoles on drug-resistant C.albicansThe results of checkerboard micro liquid-based dilution method showed that,in the combination of monomer components in KBN lotion,berberine/thymol,berberine/eugenol,palmatine/eugenol,and thymol/carvacrol has a synergistic effect against sensitive C.albicans 98001(FICI respectively:0.5,0.5,0.5,0.38);the last three groups had a synergistic effect against drug-resistant C.albicans 901(FICI respectively:0.5,0.38,0.31).In the combination of monomer components in KBN lotion and azoles,eugenol/fluconazole,eugenol/miconazole nitrate,thyme/miconazole nitrate,and carvacrol/miconazole nitrate had a synergistic effect against drug-resistant C.albicans 901(FICI respectively:<0.27,0.5,0.38,0.25).3.4 Study on KBN lotion reversed drug-resistant C.albicans resistance to fluconazoleThe results showed that KBN lotion improved the sensitivity of drug-resistant C.albicans 901 to fluconazole under the constant stimulation and induction of(1/2 MIC,MIC,2 MIC)KBN lotion,and MIC was reduced from 1024 μg/μL to 256 μg/mL.Drug-resistant C.albicans 901 within the F28 generation did not develop resistance to KBN lotion.Conclusion1 KBN lotion exerts the efficacy of inhibiting drug-resistant C.albicans through multiple pathwaysKBN lotion significantly inhibits the growth of drug-resistant C.albicans.The better anti-fungal effect and the shorter sterilization time with the higher concentration of KBN lotion,and its effect is better than that of Jieeryin xiye.Its anti-fungal mechanisms may include:the inhibition of drug-resistant C.albicans biofilm formation,hyphae growth,and adhesion;down-regulating the expression level of ERG11 gene in drug-resistant C.albicans to inhibit ergosterol synthesis;reducing the overexpression of drug-resistant efflux pump genes(CDR2 and MDR1)and the efflux hyperfunction of the efflux pumps(Cdr1p,Cdr2p,and Mdrlp).2 KBN lotion improves vaginal inflammation in VVC model animals caused by drugresistant C.albicans and its relation to the Dectin-1 signaling pathwayKBN lotion improved vaginal inflammation in VVC model mice and showed a good anti-inflammatory effect.Its anti-inflammatory mechanism may be related to reducing inflammatory factors(TNF-α,IL-1β,and IL-6)and inflammasome NLRP3 secretion by inhibiting the Dectin-1 signaling pathway(Dectin-1/Syk/PLCy-2/CARD9/NF-κB).3 KBN lotion has the potential advantages of multi-component synergy and improving the sensitivity of drug-resistant C.albicansThe pharmacodynamic substances against drug-resistant C.albicans in KBN lotion include berberine,palmatine,coptisine,thymol,carvacrol,and eugenol.Between some of the above monomer components,some monomers and azoles have a synergistic effect against drug-resistant C.albicans,and KBN lotion can improve the sensitivity of drug-resistant C.albicans to fluconazole.In conclusion,KBN lotion can improve the symptoms of vaginal inflammation in model animals caused by drug-resistant C.albicans by anti-fungal and anti-inflammatory pathways. |
PDF Full Text Request