The Effect To Berberine Hydrochloride On The Morphology And Key Gene Of Candida Albicans Biofilm

Objective:In recent years,the drug resistance caused by Candida Albicans biofilm has become increasingly serious.Studies have shown that the biofilm formation is an important factor in the growing infectious and drug resistance of the fungi.At present,a large amount of antifungal drugs have been used to treat such infections,which further increases the drug resistance.Because of its wide antifungal spectrum,obvious antifungal effect and low drug resistance,traditional chinese medicine has become an important research content of anti-infection treatment.This experiment choose berberine hydrochloride which is the major active constituent of rhizoma coptidis as the major means of intervention drug,to explore the effects and molecular regulation mechanism of Candida albicans biofilm formation.Then provide experimental basis for the selection of berberine hydrochloride auxiliary for prevention and treatment of clinical Candida Albicans biofilm infection.Methods:1.Detecting the minimum inhibitory concentration(MIC)that the berberine hydrochloride inhibits the growth of Candida albicans planktonic cell using microdilution method.Constructing biofilm model of Candida albicans in vitro,then drawing the growth curve of the biofilm using XTT experiment and observing the dynamic process of Candida albicans hyphal formation by hochest,in order to determine the formation time of hyphal and biofilm.2.Using XTT experiment and confocal laser scanning microscopy to detect the fungal metabolism and the micromorphology of Candida albicans biofilm under the intervention of the drugs at the high,medium and low concentration(4MIC,MIC,1/4MIC)of berberine hydrochloride,then study the effect of drug inhibitory for Candida albicans biofilm formation.3.Using hochest test to observe the hyphal formation of Candida albicans under the high,medium and low concentration(4MIC,MIC,1/4MIC)of berberine hydrochloride,then study the effect of drug inhibitory for Candida albicans hyphal5 formation.And further verify the relationship between the hyphal and biofilm formation.4.Using Reverse Transcription-Polymerase Chain Reaction to detect the expression of relative genes(Efg1,HWP1,ECE1,ALS1)in hyphal formation under the high,medium and low concentration,in order to research the regulation molecular mechanism of inhibition of berberine hydrochloride on Candida albicans biofilm formation.Results:1.Berberine hydrochloride has a significant inhibition effect on the Candida albicans planktonic cell,and the MIC of berberine hydrochloride is 32?g/mL.The study finds that the formation time of early biofilm,medium biofilm and mature biofilm are 0-6h,6-24 h,and 24-48 h respectively,and the time of hyphal formation is4-6h.2.For ATCC10231 and CA2119 biofilm at 4h and 6h,compared with control group,there is an obvious effect on impacting the cell activity in the biofilm,destroying the microstructure and decreasing the thickness of the biofilm with berberine hydrochloride at both 128?g/mL and 32?g/mL,the difference is significant(P < 0.05).But berberine hydrochloride at 8?g/mL only has an obvious inhibitory effect on ATCC10231,the difference is significant(P<0.05),and there is no distinct effect on CA2119.Compared with 4?g/mL fluconazole,the inhibition effect of berberine hydrochloride at 128?g/mL is better,the inhibition effect of berberine hydrochloride at 32?g/mL is weaker,the difference is significant(P < 0.05).The inhibition effect of berberine hydrochloride on the fungal metabolism and the thickness of Candida albicans at both 128?g/mL and 32?g/mL on ATCC10231 are better than CA2119,so the study shows that the inhibition effect of berberine hydrochloride on ATCC10231 is better than CA2119.At the same time,berberine hydrochloride is more effective in inhibiting the 6h biofilm of ATCC10231,showing that the biofilm of 6h may be the suitable effective time in inhibiting the fungal metabolism.3.For the yeast cells of ATCC10231 and CA2119,compared with control group,6berberine hydrochloride at 128?g/mL and 32?g/mL can significantly inhibit the yeast-hyphal phase transition of Candida albicans,the cell is less and hyphal formation is blocked.But there is no obvious inhibitory effect of berberine hydrochloride at 8?g/mL.4.For ATCC10231,compared with control group,the expression of hyphal formation key genes(Efg1,HWP1,ECE1 and ALS1)in biofilm formation can be depressed by berberine hydrochloride at 128?g/mL and 32?g/mL,and the difference is significant(P<0.05),but there is no distinct effect of berberine hydrochloride at8ug/mL.For the biofilm of 4h,compared with 4?g/mL fluconazole,the inhibition effect of berberine hydrochloride at 128?g/mL is better than fluconazole at 4?g/mL,and the difference is significant(P<0.05).For CA2119,compared with control group,the expression of hyphal formation key genes(Efg1,HWP1,ECE1 and ALS1)at 6h can be depressed by berberine hydrochloride at 128?g/mL,and the difference is significant(P<0.05),the expression of genes HWP1 and ECE1 in biofilm formation can be depressed by berberine hydrochloride at 32?g/mL and 8?g/mL.For the CA2119 biofilm at 4h,there is no distinct effect of berberine hydrochloride at128ug/mL,and the expression of genes Efg1 and ALS1 in biofilm formation can be depressed by berberine hydrochloride at 32?g/mL,the expression of genes HWP1 and ECE1 in biofilm formation can be depressed by berberine hydrochloride at 8?g/mL,and the difference is significant(P<0.05).Conclusion:1.The high concentration and the medium concentration berberine hydrochloride can inhibit the biofilm formation by killing cell inside the biofilm membrane,destroying complete structure on Candida albicans biofilm formation,and the mechanism of inhibition effect of berberine hydrochloride on the Candida albicans biofilm formation may be related to the hyphal formation.By inhibiting the genes(Efg1,HWP1,ECE1 and ALS1)expression of transition of hyphal formation,so as to inhibit the biofilm formation.2.Similar to standard strains,berberine hydrochloride have some effects on clinical strains in the inhibition of morphology and the expression of key genes,butthe inhibition effect was weaker than standard strains,showing that the intervention effect of berberine hydrochloride in clinical strains still exist many problems,need further research with a larger samples,and the drug resistance may enhance the body's resistance to other antifungal drugs.